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Expert help when you need it- Q1:250 words Summarize the following research paper and comment on the direction they could go next! (your own Idea)--> It means What direction should the research go/n nature COMMUNICATIONS ARTICLE https://doi.org/10.1038/s41467-021-21047-0 OPEN Check for updates Activatable polymer nanoagonist for second near- infrared photothermal immunotherapy of cancer Yuyan Jiang, Jiaguo Huang, Cheng Xu¹ & Kanyi Pu① 1,2区 Nanomedicine in combination with immunotherapy offers opportunities to treat cancer in a safe and effective manner; however, remote control of immune response with spatiotemporal precision remains challenging. We herein report a photothermally activatable polymeric pro- nanoagonist (APNA) that is specifically regulated by deep-tissue-penetrating second near- infrared (NIR-II) light for combinational photothermal immunotherapy. APNA is constructed from covalent conjugation of an immunostimulant onto a NIR-II semiconducting transducer through a labile thermo-responsive linker. Upon NIR-II photoirradiation, APNA mediates photothermal effect, which not only triggers tumor ablation and immunogenic cell death but also initiates the cleavage of thermolabile linker to liberate caged agonist for in-situ immune activation in deep solid tumor (8 mm). Such controlled immune regulation potentiates sys- temic antitumor immunity, leading to promoted cytotoxic T lymphocytes and helper T cell infiltration in distal tumor, lung and liver to inhibit cancer metastasis. Thereby, the present work illustrates a generic strategy to prepare pro-immunostimulants for spatiotemporal regulation of cancer nano-immunotherapy. 1 School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore, Singapore. 2 Division of Chemistry and Biological Chemistry, School of Physical and Mathematical Sciences, Nanyang Technological University, Singapore, Singapore. email: kypu@ntu.edu.sg NATURE COMMUNICATIONS | (2021)12:742 | https://doi.org/10.1038/s41467-021-21047-0 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21047-0 mmunotherapy that boosts host immune system to fight against tumor has revolutionized cancer treatment 1,2. How- ever, single model immunotherapy often suffers from limited response rate and occasional immune-related adverse effects (irAEs) such as cytokine storm, hematopoietic system dysfunc- tion, and organ failure³. Nanomedicine thus has been brought into cancer immunotherapy in the hope to address these critical concerns in view of its tunable physicochemical properties to reinforce drug-immune cell interactions, and optimal pharma- cological profiles (e.g., biodistribution and retention) of immu- notherapeutic molecules 4-6. More importantly, nanomedicine serves as an assembly platform to allow additional therapeutic modalities (e.g., chemotherapy, phototherapy, radiotherapy, etc.) to be seamlessly integrated with immunotherapy to enhance therapeutic efficacy7-11. With the promise to improve immune response at no expense of breaking the systemic immune toler- ance, cancer nano-immunotherapy has been recently under extensive development. Further enhancement in the specificity of cancer immu- notherapy to facilitate its clinical translation mainly lies in the development of smart immunotherapeutic nanoagents with controlled activation 12-14. Till now, immunotherapeutic nanoa- gents have been programmed to unleash therapeutic action in response to biochemical indexes or cancer biomarkers such as pH, reactive oxidative species, and enzymes 15-18. For instance, human interleukin-15 super-agonist complex was engineered with disulfide-containing cross-linker for activatable release in response to T-cell receptor activation so as to improve therapeutic window of adjuvant cytokine therapy19. As another example, programmed cell death protein 1 antibody was specifically con- jugated to magnetic nanoclusters via pH-responsive benzoic- imine bond, leading to selective activation in the acidic intratu- moral microenvironment for potentiation of adoptive T-cell therapy 20. However, owing to their reliance on the difference of biomarkers at basal and pathological levels, the bioavailability and regional selectivity of immune activation for such immunother- apeutic nanoagents remain to be improved. In contrast to endogenous biomarkers, external stimuli are independent of the physiological environment and thus hold potential for more precise spatiotemporal and dosage control over immune regulation 12,21. Near-infrared light (NIR-I, 650-950 nm) is a popular external stimulus for the construction of smart nanomedicine owing to its minimal invasiveness and easy operation²². In particular, NIR-I light-responsive nanoagents have been developed to serve as the signal transducers to convert incident photons into regulatory signals, such as singlet oxygen to trigger the activation of indoleamine 2,3-dioxygenase 1 inhibitor, or high-energy emission to liberate CpG21,23. Superior to NIR-I light, second NIR light (NIR-II, 1000-1300 nm) has been recently revealed to possess even better biological transparency and fur- ther ameliorated phototoxicity with a lower maximum permis- sible exposure limit 24. However, few NIR-II nanotransducers are available, which include gold nanostructures25, transition metal- based nanoparticles26, and naphthalocyanine derivatives27. Recently, semiconducting polymer nanoparticles (SPNs) com- posed of highly л-conjugated backbones have formed a promising class of NIR-II nanoagents 28,29. With their excellent photo- thermal performance and intrinsically benign compositions, SPNs have been exploited for deep-tissue molecular photoacoustic imaging³ photothermal therapy (PTT)³², and photothermal ferrotherapy33. However, the integration of SPNs with NIR-II light for spatiotemporal photoregulation of immunotherapy has yet to be explored. 30,31 We herein report the synthesis of an activatable polymer nanoagonist (APNA) for NIR-II light-regulated photothermal immunotherapy of cancer (Fig. 1). APNA is composed of a NIR- 2 II light-absorbing semiconducting polymer backbone as photo- thermal transducer, conjugated with a potent toll-like receptor type 7 and 8 (TLR7/8) agonist (Resiquimod: R848) as the immunostimulant through a thermolabile cleavable linker (2,2'- azobis [2-(2-imidazolin-2-yl)propane]: VA-044) (Fig. 1a). Mainly acting on antigen-presenting cells (APCs) such as dendritic cells (DCs), R848 helps upregulate secretion of crucial proin- flammatory cytokines and enhance maturation or polarization of APCs, priming T lymphocytes³4. Upon focal NIR-II photo- irradiation, APNA mediates photothermal effect to directly ablate tumor, and elicit immunogenic cell deaths (ICDs) of cancer cells to promote antitumor immunity; it also in situ triggers the cleavage of thermolabile linker at the tumor site to activate the TLR7/8 agonist so as to further potentiate antitumor immune response (Fig. 1b). Such an APC-mediated spatiotemporal potentiation of cancer immunotherapy enables complete eradi- cation of the primary tumor in deep tissue (~8 mm) and efficient inhibition of both distal tumor and lung metastasis without eli- citing obvious systemic adverse effects. Furthermore, the under- lying mechanisms of photothermal activation of immune response along the gradient of photothermal depth and in situ photothermal temperature in the deep beds of tumor are unveiled, providing guidelines for the development of photo- thermal immunotherapy. Results Synthesis and in vitro characterization. To obtain the NIR-II- absorbing semiconducting polymer precursor pBODO-Br, Stille polycondensation was used to copolymerize three monomers: 4,8-bis[5-bromo-4-(2-octyldodecyl)-2-thienyl]-benzo[1,2c:4,5c']bis [1,2,5]thiadiazole (BBT), 2,5-bis(6-bromohexyl)-3,6-bis(5-bro- mothiophen-2-yl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (DPP- Br), and (4,8-bis((2-ethylhexyl)oxy)benzo[1,2b:4,5-b'] dithiophene- 2,6-diyl)bis(trimethylstannane) (OT) (Fig. 2a). Because of the strong-electron withdrawing of BBT and the electron-rich OT, PBODO-Br has a charge transfer backbone and narrowed band gap, showing strong absorption in the NIR-II window (Supple- mentary Fig. 1). Gel permeation chromatography analysis indi- cated that pBODO-Br had a number average molecular weight (Mn) of 9826 (Supplementary Fig. 2). pBODO-Br was further transformed to pBODO-N3 through substitution of bromide with azide for post-functionalization (Supplementary Fig. 3). To con- jugate the thermo-responsive linker VA-044 with the immuno- agonist R848, VA-044 was first modified with 7-iodoheptanoic acid, followed by Steglich esterification reaction with R848 to afford caged agonist VR (Supplementary Figs. 4, 5). A bifunctional poly(ethylene glycol) (PEG, Mw=2000) with one propargyl terminal and the other amine terminal (alkyne-PEG-NH2) was synthesized and linked to VR via EDC/NHS coupling to obtain alkyne-PEG-VR conjugate (Supplementary Figs. 6, 7). Thereafter, PBODO-N3 was grafted with alkyne-PEG-VR to afford semi- conducting pro-agonist pBODO-PEG-VR through click reaction (Supplementary Fig. 8). The amphiphilicity of pBODO-PEG-VR allowed its spontaneous assembly in aqueous solution to form nanoparticles termed as APNA (Fig. 1a). To prepare the control nanoparticle without pro-agonist termed as APNC, pBODO-N3 was conjugated with alkyne terminated PEG (pBODO-PEG), fol- lowed by self-assembly in aqueous solution. Optical and colloidal properties of APNA were studied and compared with APNC. Both APNA and APNC had similar absorption spectra with maxima at 690 nm in NIR-I window and 1060 nm in NIR-II window (Fig. 2b), showing that conjugation of pro-agonist had negligible influence on NIR-II light-harvesting property. Whereas dynamic light scattering (DLS) indicated a larger hydrodynamic size of APNA (71 nm) than APNC (48 nm) NATURE COMMUNICATIONS | (2021)12:742 | https://doi.org/10.1038/s41467-021-21047-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21047-0 a N C2H5 C4H9 C2H5 C4H9 R = N=N -N S 0.5 n C8H17 C10H21 0.5 NN C2H5 C4H9 C₂H C4H9 NIR-II Photothermal Unit b 0 Photothermal depth (mm) 9 4 7 8 PBODO-PEG-VR i. NIR-II photoirradiation O Primary Tumor Tumor temperature (°C) 50 45 43 42 41 Self- assembly ARTICLE PEG Thermolabile linker Caged agonist ΑΡΝΑ NH₂ ii. Photothermia Immunogenic cell death Photothermal activation TAAS DAMPS (HMGB1, etc.) Inhibition of lung metastasis v. T cell infiltration iv. T cell activation CD8+ T Cell Distant Tumor Abscopal Effect CD4+ T Cell Lymph node iDC Activated agonist iii. DC Maturation Migration MDC Fig. 1 Scheme of APNA-mediated NIR-II photothermal immunotherapy. a Chemical structure of pBODO-PEG-VR and preparation of APNA. b Mechanism of antitumor immune response by APNA-mediated NIR-II photothermal immunotherapy. TAAS tumor-associated antigens, DAMPs damage-associated molecular patterns, iDC immature DC, mDC mature DC, HMGB1 high-mobility group box 1 protein. (Fig. 2c), probably owing to the presence of hydrophobic agonist molecules. Transmission electron microscopy (TEM) further confirmed spherical morphology for both APNA and APNC (Fig. 2c). In addition, zeta potential measurement indicated a relatively neutralized surface charge of APNA (-13 mV) in contrast with APNC (-29 mV) (Supplementary Fig. 9a). Both nanoparticles showed negligible size change during storage in aqueous solutions for 2 months (Supplementary Fig. 9b), suggesting their excellent colloidal stability. According to high performance liquid chromatography (HPLC) calibration curve, the drug loading capacity of APNA was ca. 5.3%, suggesting that each pBODO-PEG-VR molecule contained ~3.3 equivalents of R848 pro-drug. Photothermal property of APNA was evaluated and compared with APNC. Upon continuous NIR-II (1064 nm) photoirradia- tion, both APNA and APNC induced significant temperature rise of aqueous solution (Fig. 2d). After irradiation for 6 min, the maximum solution temperatures of APNA and APNC nanopar- ticles were 78 and 76 °C, respectively. Such phenomenon implied the negligible impact of agonist conjugation on the photothermal transduction capability of semiconducting backbone. Indeed, APNA and APNC had similar photothermal conversion efficiency as high as 84.4% at 1064 nm (Supplementary Fig. 10). In addition, negligible changes in maximal solution temperatures were observed for both APNA and APNC nanoparticles throughout five heating and natural cooling cycles, suggesting their excellent photostability as photothermal agents. Photothermal activation of pro-agonist from APNA was evaluated and analyzed by HPLC (Fig. 2e, f). Without photo- irradiation, no elution peak related to free R848 (TR = 17.9 min) from APNA nanoparticles could be measured (Fig. 2f). After continuous photoirradiation of APNA solution for 10 min, an elution peak at 24.7 min assigned to the released agonist was observed. Such product from the photothermal cleavage was further confirmed by liquid chromatography-mass spectrometry (LCMS) and proton nuclear magnetic resonance spectroscopy ('H NMR) (Supplementary Figs. 11, 12). Through subsequent hydrolysis by esterase, the parent R848 was able to be liberated from the photothermally activated pro-agonist in an intact form, which was validated by both HPLC profiles and LCMS (Fig. 2f, Supplementary Fig. 13). Thereafter, the relationship between photothermal temperature and photothermal activation rate was investigated (Fig. 2g). When the photothermal temperature was kept at 37°C for 20 min under photoirradiation, a maximal agonist activation ratio of 8.5% was achieved. Whereas such ratio was dramatically promoted to 74% when photothermal tempera- ture was elevated to 45 °C or 55 °C and maintained for 20 min. Notably, such high activation ratio was achieved in 5 min with NATURE COMMUNICATIONS | (2021)12:742 | https://doi.org/10.1038/s41467-021-21047-0 | www.nature.com/naturecommunications 3 ARTICLE Absorption a HOOC N NN NEN N ii N=N NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21047-0 VA-044 VCOOH VR COOH COOH Br C2H5 C4H9 C2H5 C4H9 OT C8H17 C10H21 + N...N S Br S. C8H17 C10H21 DPP-Br Br C2H5 CH9 N NH2 !!! Болон Alkyne-PEG-VR S. N N C2H5C4H9 iv NN -Br N..N C8H17 SC8H17 C10H21 C10H21 N.. N S C10H21 BBT C2H5 C4H9 pBODO-Br Br H₂N C2H5C4H9 HK 0. S0.5 n C2H5 C4H9 №3 vi vii C2H5 C4H9 PBODO-PEG-VR ΑΡΝΑ C8H11C10H21 -$0.5 C2H5 C4H9 PBODO-N3 N3 S 0.5 n C2H5 C4H9 b 0.8 ΑΡΝΑ APNC 0.6 0.4 0.2 0.0 00 e 600 800 Wavelength (nm) 1064 nm C 25 viii ix PBODO-PEG APNC d 100- APNC ΑΡΝΑ 80 Number (%) ΑΡΝΑ 20 APNC 15 10 5 APNA APNC 50 nm Temperature (°C) 0 N% 60 40 20 1000 1 10 100 1000 10000 Diameter (nm) Caged agonist R848 APNA - laser ΑΡΝΑ Activated agonist H₂N APNA + laser H₂N R848 Esterase H₂N APNA + laser + esterase 0 10 20 Time (min) g 0.8 Drug Release Ratio 0.6 0.4 0.2- 0 720 1440 2160 2880 3600 Time (s) 37 °C 45 °C - 55 °C 0.0 0.0 0 5 10 15 Laser irradiation time (min) 20 Fig. 2 In vitro characterization of APNA. a Synthetic routes of APNA and APNC. (i) 7-iodoheptanoic acid, sodium hydride (NaH), dry tetrahydrofuran (THF), 25 °C, 2d. (ii) R848, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), 4-dimethylaminopyridine (DMAP), dry acetonitrile (ACN), 25 °C, 3 d. (iii) alkyne-PEG-NH2, EDC, N-hydroxysuccinimide (NHS), dry THF, 25 °C, 2d. (iv) Pd2(dba)3, tri(o-tolyl)phosphine, chlorobenzene, 100 °C, 2 h. (v) sodium azide, dimethylformamide (DMF)/THF, 25 °C, 2d. (vi) alkyne-PEG-VR, CuBr, N,N,N',N",N"-pentamethyldiethylenetriamine (PMDETA), dry THF, 25 °C, 2d. (vii) aqueous self-assembly of pBODO-PEG-VR. (viii) methoxy-PEG-alkyne, CuBr, PMDETA, dry THF, 25 °C, 2d. (ix) aqueous self-assembly of pBODO- PEG. b Absorption spectra of APNA and APNC in 1× PBS solution. c DLS profiles of APNA and APNC nanoparticles. Inset: TEM images of APNA and APNC nanoparticles. d Photostability studies of APNA and APNC ([pBODO] = 20 μg mL −¹) by photothermal heating (1064 nm photoirradiation, 1 W cm-2, 6 min) and cooling (natural cooling, 6 min) cycles. e Schematic illustration of photothermally triggered agonist release from APNA and subsequent hydrolysis by esterase. f HPLC analysis of photothermally triggered agonist release from APNA ([pBODO] = 50 μg mL−1). 1064 nm photoirradiation: 1 W cm-2, 10 min. g Relationship of photothermal activation ratio and different photothermal temperatures ([pBODO] = 50 µg mL−1). The photothermal temperature was controlled by switching power density of photoirradiation. Data were expressed as mean ± SD. Error bars indicated standard deviations of three independent measurements. 4 NATURE COMMUNICATIONS | (2021)12:742 | https://doi.org/10.1038/s41467-021-21047-0 | www.nature.com/naturecommunications d NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-21047-0 C a DAPI Lysosome SPN Merge DAPI Lysosome SPN Merge 120 APNAF PBS APNCF 20 μm - Cell Viability (%) 100- 80 60 60 40 60 APNC - Laser APNA Laser 40 APNC + Laser APNA + Laser 20 20 μm 0 5 10 ARTICLE 20 50 Concentration (μg mL-1) e PBS R848 APNC - Laser R848 Incubation 105 4.12% 9.57% 105 12.3% 64.1% 105 2.89% 13.7% CD80 CD86 ↑t 104- 104- 104- iDC MDC 103- 103- APNA-Laser 103- 0 0 0 CD80 D CD861 80.0% -103 6.27% 21.3% 2.32% 81.4% -103 1.97% -103 iDC mDC -103 0 103 104 105 -103 0 103 104 105 -1030 103 104 105 APNC+Laser APNC + Laser APNA - Laser APNA + Laser CD80 105- 3.13% CD86 20.2% 105- 10.6% 30.1% 105 4.35% 57.5% iDC mDC APNA+Laser CD80 CD86 iDC mDC CD80-FITC 104 104- 104- 103- 103- 103- 0 72.7% 3.94% -103 0 -103 0 56.7% 2.52% 34% 4.18% -103 -103 0 103 104 105 -103 0 103 -103 0 103 104 105 → CD86-PE 104 105 Fig. 3 In vitro APNA-mediated photothermal immunotherapy. a, b Confocal fluorescence images of cellular internalization (24 h) of APNAF and APNCF ([pBODO] = 10 μg mL-1) in 4T1 cancer cells a and BMDCs b. APNA and APNC were labeled with NCBS (2.5 w/w%). Blue fluorescence indicated nuclei staining by 4', 6-diamidine-2'-phenylindole dihydrochloride (DAPI). Green fluorescence indicated lysosomes staining by LysoTracker Green DND-26. Red fluorescence denoted as SPN channel indicated NIR fluorescence from APNAF or APNCF. Experiments were performed in triplicate with similar results. c Cell viability assay of 4T1 cells at 12 h after treatment with APNC or APNA at different concentrations with or without 1064 nm photoirradiation (1 W cm-2, 6 min) (n = 3). d Schematic illustration of pre-photothermal activation of APNA and subsequent in vitro stimulation of iDC. e Flow cytometry analysis of BMDC maturation (gated on CD11c+ DCs) at 48 h after various treatments. [R848] = 4.4 µg mL−1; [pBODO] = 10 μg mL −1; 1064 nm photoirradiation, 1W cm−², 6 min. Data were expressed as mean ± SD. photothermal temperature at 55 °C, whereas extended to 10 min at 45 °C. These results indicated the agonist could be selectively activated by photothermal heating mediated by the backbone of APNA. In vitro photothermal ablation and DC maturation. Cellular uptake of APNA and APNC was evaluated on 4T1 murine breast cancer cells and bone marrow-derived DCs (BMDCs). A fluor- escent dye (silicon 2,3-naphthalocyanine bis(trihexylsilyloxide)) (NCBS) was doped into nanoparticles (2.5 w/w%) to obtain fluorescent derivatives (APNA and APNCF) (Supplementary Figs. 14, 15). After treating 4T1 cells with APNAF or APNCF for 24 h, strong NIR fluorescence was observed from cellular plasma (Fig. 3a), indicating effective endocytosis of both nanoparticles in 4T1 cells. Similar uptake was detected for BMDCs (Fig. 3b, Supplementary Fig. 16). Furthermore, the nanoparticle signal overlapped well with the green fluorescence from lysosome staining, suggesting that endocytosed nanoparticles mainly resi- ded in lysosomes, wherein TLR7/8 was highly expressed 35,36 Without photoirradiation, both APNA and APNC induced neg- ligible cytotoxicity to 4T1 cells (Fig. 3c). However, with NIR-II photoirradiation, both nanoparticles triggered significant cell death relative to the control group. For instance, at 50 μg mL the cell viability of APNA and APNC treated-group decreased to 24% and 25%, respectively. ' The ability of APNA to photothermally trigger in vitro immune activation was investigated on DCs, a crucial species of APCs whose maturation impacts the activation of both innate and adaptive immunity37. After photoirradiation of APNA or APNC solution for 10 min, the solutions were transferred to the culture media of immature BMDCs, followed by detection of their maturation by flow cytometry analysis on the expression of the costimulatory molecules CD80 and CD86 (Fig. 3d, Supplementary Fig. 17). The post-photoirradiated APNA triggered upregulation of CD80 and CD86 (57.5%), which was 1.9-fold and 2.8-fold higher than unirradiated APNA (30.1%) or the post- photoirradiated control (APNC) (20.2%) (Fig. 3e). Furthermore, the DC maturation induced by APNA after photothermal activation was similar to free R848 at 4.4 µg mL-¹. Consistently, mixed lymphocyte reaction indicated the superior T-cell stimula- tion capacity of pre-activated APNA-treated DCs, as revealed by the higher percentage of CD3+CD8+ T cells and elevated IFN-y production relative to other groups (Supplementary Fig. 18). These results show that the immune-stimulating ability of APNA was effectively activated by NIR-II photoirradiation. NATURE COMMUNICATIONS | (2021)12:742 | https://doi.org/10.1038/s41467-021-21047-0 | www.nature.com/naturecommunications 5See Answer
- Q2: TO DO - Need to Summarize the following research paper and comment on the direction they could go next or how to make the paper better! (your own Idea) WORD LIMIT - 250 ( altogether), MLA/n frontiers in Pharmacology REVIEW published: 03 February 2021 doi: 10.3389/fphar.2021.601626 Check for updates Biodegradable Polymeric Nanoparticles for Drug Delivery to Solid Tumors Agnese Gagliardi¹t, Elena Giuliano ¹t, Eeda Venkateswararao², Massimo Fresta¹, Stefania Bulotta 1*, Vibhudutta Awasthi²* and Donato Cosco 1* 1 Department of Health Sciences, University "Magna Græcia" of Catanzaro, Catanzaro, Italy, 2 Department of Pharmaceutical Sciences, The University of Oklahoma Health Sciences Center, Oklahoma City, OK, United States OPEN ACCESS Edited by: Santiago Gómez-Ruiz, Rey Juan Carlos University, Spain Reviewed by: Kui Luo, Sichuan University, China Xiaowei Zeng, Sun Yat-Sen University, China *Correspondence: Stefania Bulotta bulotta@unicz.it Vibhudutta Awasthi vawasthi@ouhsc.edu Donato Cosco donatocosco@unicz.it These authors have contributed equally to this work Specialty section: This article was submitted to Experimental Pharmacology and Drug Discovery, a section of the journal Frontiers in Pharmacology Received: 01 September 2020 Accepted: 04 January 2021 Published: 03 February 2021 Citation: Gagliardi A, Giuliano E, Venkateswararao E, Fresta M, Bulotta S, Awasthi V and Cosco D (2021) Biodegradable Polymeric Nanoparticles for Drug Delivery to Solid Tumors. Front. Pharmacol. 12:601626. doi: 10.3389/fphar.2021.601626 Advances in nanotechnology have favored the development of novel colloidal formulations able to modulate the pharmacological and biopharmaceutical properties of drugs. The peculiar physico-chemical and technological properties of nanomaterial-based therapeutics have allowed for several successful applications in the treatment of cancer. The size, shape, charge and patterning of nanoscale therapeutic molecules are parameters that need to be investigated and modulated in order to promote and optimize cell and tissue interaction. In this review, the use of polymeric nanoparticles as drug delivery systems of anticancer compounds, their physico-chemical properties and their ability to be efficiently localized in specific tumor tissues have been described. The nanoencapsulation of antitumor active compounds in polymeric systems is a promising approach to improve the efficacy of various tumor treatments. Keywords: surfactants, PEG, polymeric nanoparticles, passive targeting, cancer INTRODUCTION Cancer is the second leading cause of death in the world, and was responsible for approximately 9.6 million deaths in 2018 (Bray et al., 2018). Over the next 20 years, the number of new cases is estimated to increase by about 70% (Siegel et al., 2017). Cancer therapy is considered a multidisciplinary challenge requiring close collaboration among clinicians, biologists, and biomedical engineers (Danhier et al., 2010). Current cancer treatments include surgery, radiation, and chemotherapy, but the effects of these procedures may cause damage to normal as well as tumoral cells. The resultant systemic toxicity and adverse effects greatly limit the maximum tolerated dose of anti-cancer drugs, and thus restrict their therapeutic efficacy. In particular, surgery together with radiotherapy are the first choice used for local and non-metastatic cancers, while anti- cancer drugs (chemotherapy, hormone, and biological therapies) are the treatments currently employed in metastatic cancers and adjuvant therapies (Tran et al., 2017). The toxicity of conventional chemotherapeutic drugs, as well as the indiscriminate destruction of healthy cells and the development of multidrug resistance, are the motivating thrust behind research on novel targeted treatments (Pérez-Herrero and Fernández-Medarde, 2015; Tran et al., 2017). The main challenge is to improve the selectivity of anticancer drugs for tumor cells and the tumor microenvironment, while sparing healthy cells and tissues. In this context, a promising approach is the targeting of tumor tissue by nanomedicine-based therapeutics (Oerlemans et al., 2010). These formulations are made up of submicrometer-sized carriers containing the active compound(s), which are able to selectively diagnose and treat tumors by suitable targeting vectors, thus improving the therapeutic index and the pharmacokinetic profile of the anticancer drugs that are delivered. Frontiers in Pharmacology | www.frontiersin.org 1 February 2021 | Volume 12 | Article 601626 Gagliardi et al. Polymeric Nanoparticles as Antitumor Nanomedicine Polymeric Nanoparticles Solid Tumor Drug Targeting Natural Synthetic Size Biopolymers Surface Polydispersity Preparation Nanosphere Polymeric Shell Chemisti Properties Oncological Applications Polymeric Matrix Drug Clinical and preclinical investigation Immunotherapy FIGURE 1 | Overview of the main features of polymeric nanoparticles. Nanocapsule Polymeric Nanoparticles Future perspectives Nanocarriers can retain multiple therapeutic agents not only to enhance their therapeutic effect on a synergestic or additive basis, but also to overcome acquired resistance to single chemotherapeutic drugs. Many tumors develop chemo- resistance through many mechanisms, including induction of the drug efflux rate or the downregulation of uptake mechanisms (Mansoori et al., 2017). Nanoparticulate formulations can overcome this limitation by providing an alternative pathway of cellular internalization. Currently, several therapeutic nanoparticle platforms are being investigated for targeted cancer treatment, including lipid-based, polymer-based, inorganic, viral, and polymer-drug conjugated systems. In the past two decades, over 20 nanotechnology-based therapeutic products have been approved for clinical use. Among these products, liposomal systems and polymer-drug conjugates are two of the most important groups, and many other formulations are under clinical investigation, including chemotherapy, hyperthermia, radiation therapy, gene or RNA interference (RNAi) therapy, and immunotherapy (Wicki et al., 2015). Nanocarriers have unique features such as their nanometric size, high surface area-to-volume ratio, favorable drug release profiles and targeting features which can promote their preferential accumulation in tumor tissues (Wicki et al., 2015). Most nanosystems for the treatment of solid tumors are administered systemically and accumulated in the tumor tissues through the enhanced permeability and retention (EPR) effect, which is generally thought to be the result of leaky tumor vasculature and poor lymphatic drainage (Maeda, 2015). However, this interpretation of EPR-dependence is simplistic, because the biodistribution of systemically administerd nanosystems can be influenced by multiple biological factors, including interaction with plasma proteins, blood circulation time, extravasation, penetration of tumor tissue, and cancer cell uptake (Shi et al., 2017). Modification of the surfaces of the nano-systems-which are able to confer specific targeting properties or stimuli-sensitive responses—also affect their overall distribution. Much of our current knowledge regarding the in vivo behavior of nanoparticulate systems is based on data obtained from animal models. But relatively few investigations have correlated the obtained data in order to determine whether and how the safety and the efficacy of nanoparticles in humans can be better predicted by using these animal models (Hrkach et al., 2012; Zuckerman et al., 2014). There also exist a number of scientific articles which focus on specific aspects and applications concerning the development of polymeric nanoparticles. Frontiers in Pharmacology | www.frontiersin.org 2 February 2021 | Volume 12 | Article 601626 Gagliardi et al. Macrophage # Cancer Cells Red Cells Polymeric Nanoparticles as Antitumor Nanomedicine Endothelial Cells Fibroblast # Collagen Fibers Physiological Environment Pericyte FIGURE 2 | Differences between a physiological and a tumor environment. Figure generated from Servier Medical Art. Tumor Environment Therefore, this work is not intended to be a review of all the research performed in this area, but rather to provide the basic concepts and ideas related to the preparation and use of polymer- based nanoparticles as drug carriers in cancer therapy. This article offers an overview and discusses the most important findings and prospects as illustrated in Figure 1. TUMOR MICROENVIRONMENT The microenvironment of tumor tissue significantly differs from that of healthy tissue. These differences include vascular abnormalities, oxygenation and perfusion levels, pH, and metabolic status (Abadjian et al., 2017). Solid tumors are characterized by a heterogeneous population of neoplastic cells supplied by an irregular and discontinuous endothelium with large gaps between the endothelial cells, and abnormally thick or thin basement membranes where pericites are loosely attached to endothelial cells (Figure 2). The irregularity of tumor blood vessels in their distribution, diameter, density, and serpentine shape, can be the cause of poor perfusion which leads to excessive fluid extravasation (Khawar et al., 2015). The two main causes of this heterogeneity are spatial stress, resulting from rapid tumor growth, and the abnormal extracellular matrix which can compress the vessels and partially block the flow of blood [which causes the escape of plasma and a high interstitial fluid pressure (IFP)] (Jain, 2013). The IFP is highest at the center of solid tumors and decreases radially, creating a movement of fluid away from the central region of the tumor. This phenomenon contributes to a reduced transcapillary transport of therapeutic drugs as well as their scarce accumulation in the middle of the tumor (Danhier et al., 2010). The elevated IFP and associated peritumoral edema also assist in the transport of growth factors and cancer cells away from the tumor, thus favoring tumor progression, while the abnormal and disorganized tumor vasculature results in inefficient blood flow inside the tumor mass, hypoxia, and low extracellular pH (Khawar et al., 2015). Hypoxia plays a crucial role in tumor growth and metastasis through the induction of molecular signaling which is responsible for genetic instability, inflammation, immunosuppression, epithelial-mesenchymal transition and altered metabolism (Jing et al., 2019). It also confers resistance against several kinds of treatment, such as radiation, chemo-, photodynamic and immunotherapies, which require oxygen for efficacy (Jain, 2013). By virtue of the hypoxia-inducible factor-mediated pathway, hypoxia promotes angiogenesis. Oxygen can diffuse for maximum 150 μm beyond the capillary wall, which implies that when a tumor reaches a certain size (~2 mm³), a state of cellular hypoxia begins. Angiogenesis is a cellular mechanism which is upregulated in tumoral microenvironments and creates new blood vessels to further assist tumor growth by supplying oxygen and nutrients (Jászai and Schmidt, 2019). This process consists of five steps: i) endothelial cell activation, ii) basement membrane degradation, iii) endothelial cell migration, iv) new vessel formation, and v) angiogenic remodeling. In the first phase, hypoxia induces an increase of the hypoxia-inducible factor- mediated transcription of pro-angiogenic proteins such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and tumor necrosis factor-a (TNF-α) (Wigerup et al., 2016). Activated endothelium regulates the migration of endothelial cells through the extracellular matrix during vessel formation, due to the expression of a dimeric Frontiers in Pharmacology | www.frontiersin.org 3 February 2021 | Volume 12 | Article 601626 Gagliardi et al. Polymeric Nanoparticles as Antitumor Nanomedicine transmembrane integrin avß3 which interacts with the proteins of the extracellular matrix (vitronectin, fibronectin, etc.) (Demircioglu and Hodivala-Dilke, 2016). Successively, the matrix metalloproteinases synthesized by the activated endothelial cells degrade the basement membrane and the extracellular matrix. This process causes the apoptosis of the inner layer of endothelial cells, leading to the formation of a vessel lumen and remodeling of the immature vasculature, stabilized by pericytes and smooth-muscle cells. Often this step remains incomplete, resulting in irregularly-shaped, dilated, and tortuous tumor blood vessels. The angiogenic switch is the crucial phase in which a tumor changes from a non- angiogenic to an angiogenic phenotype and allows the dissemination of cancer cells throughout the body (Jászai and Schmidt, 2019). Hypoxia also results in metabolic acidosis caused by increased glycolysis (the Warburg effect); this lowers the extracellular pH to 6.0-7.0 (De Palma et al., 2017). Acidosis is also a factor in epithelial-mesenchymal transition and it synergistically contributes to tumor invasion and metastasis (Yang et al., 2016). Because of their rapid growth, tumor cells continue to exploit glycolysis as an ATP-generating pathway even when oxygen is available, lowering dependence on glucose oxidation for energy production (Fu et al., 2017). This metabolic preference is mostly due to defective mitochondrial function (Kim et al., 2009). The elevated breakdown of glucose produces large amounts of lactic acid and significant amounts of free protons (H+) which are pumped into the extracellular milieu by mechanisms involving the carbonic anhydrases IX and XII (Martinez-Outschoorn et al., 2017). The resulting pH gradients between intra- and extracellular compartments within the tumor tissue, as well as between the tumor mass and the general host tissue, are potential sources of variable and often inefficient partitioning and distribution of drugs. Exposure to chemotherapy may favor the selection of tumor-cell clones with acidic organelles, which are able to entrap the drugs, and if these organelles are part of the secretory pathway, then the drug will be transported out of the cell through exocytosis. All these factors in the tumor microenvironment contribute to multidrug resistance (MDR) phenomena (Danhier et al., 2010). POLYMERIC NANOPARTICLES Over the last decade nanoparticles have become extremely attractive for application in biology and medicine (Mogoşanu et al., 2016). They have the potential to modulate biopharmaceutical features, pharmacokinetic properties, and the therapeutic efficacy of entrapped drugs (Dang and Guan, 2020). Technically, nanoparticles are defined as being less than 100 nm, but in practice structures up to 300 nm in size are included in this category (Guo et al., 2016), and they can fall into different classes as a function of their morphology, size, composition, and physicochemical properties (Khan et al., 2019). Polymer-based nanoparticles are colloidal systems made up of natural or synthetic polymers. They furnish significant advantages over other nanocarriers such as liposomes, micelles and inorganic nanosystems, and include the feasibility of scale-up and the manufacturing process under Good Manufacturing Practices (GMP) (van Vlerken et al., 2007). Other peculiar characteristics of polymeric nanoparticles are the significant stability of polymeric nanoparticles in biological fluids along with the wide availability of various polymers, the opportunity to functionalize their surfaces and to modulate polymer degradation and the leakge of the entrapped compound(s) as a function of specific stimuli (Venkatraman et al., 2010; Goodall et al., 2015; Sarcan et al., 2018). Several chemotherapeutics have been encapsulated in polymeric delivery systems, with the aim of increasing antitumor efficacy, inhibiting metastases, and decreasing the effective dose and side effects. Polymers can encapsulate an active compound within their structure or adsorb it onto their surfaces (Masood, 2016). Langer and Folkman were the first to demonstrate the controlled release of macromolecules using polymers, which allowed the development of antiangiogenic drug delivery systems for cancer therapy (Langer and Folkman, 1976). Ideally, the polymers selected for parenteral administration must be biocompatible, biodegradable, and possess specific mechanical and physicochemical properties (Vilar et al., 2012). The first polymers used to develop polymeric nanoparticles (PNs) were non-biodegradable polymers, such as poly(methyl methacrylate) (PMMA), polyacrylamide, polystyrene, and polyacrylates. The nanosystems made up of these materials exhibited a rapid and efficient clearance, but chronic toxicity and inflammatory reactions were observed. Usually, non-degradable polymers require degradation times longer than their effective duration of application (Anju et al., 2020), whereas the degradation rate of biodegradable polymeric nanoparticles can be influenced by several parameters, including their physico-chemical properties (size, structure, molecular weight) and external factors, such as pH and temperature (Su and Kang, 2020). Although pioneering studies on polymeric nanoparticles have focused on non-degradable materials, the use of biodegradable polymers had a great impact as a consequence of their notable biocompatibility and biosafety (Kamaly et al., 2016). Biodegradable polymers include synthetic polymers such as poly(D,L-lactide) (PLA), poly(D,L-glycolide) (PLG), co-polymer poly(lactide-co-glycolide) (PLGA), polyalkylcyanoacrylates, poly-Ɛ-caprolactone. They are considered safe and a few biodegradable polymer products have been approved by the US Food and Drug Administration (FDA) as well as by the European Medicines Agency (EMA) for pharmaceutical application (Palma et al., 2018). In general, biodegradable polymeric particles show reduced systemic toxicity, are more biocompatible, and favor modulation of drug-release kinetics. They are typically degraded into oligomers and monomers, which are further metabolized and eliminated from the body via normal pathways (Ravivarapu et al., 2006; Vilar et al., 2012). Non- synthetic biodegradable polymers, which include natural polymers such as chitosan, alginate, gelatin, zein, and albumin, have also been used to prepare polymeric nanoparticles (Gagliardi et al., 2018). We will discuss commonly-used Frontiers in Pharmacology | www.frontiersin.org 4 February 2021 | Volume 12 | Article 601626 Gagliardi et al. Polymeric Nanoparticles as Antitumor Nanomedicine polymers for the preparation of drug-loaded PNs for anticancer therapy later. Biopolymers for Cancer Nanomedicine Biopolymers are one of the most important classes of biomaterials (Anju et al., 2020) and are widely used in biomedical applications because of their biocompatibility and biodegradability (Jaimes- Aguirre et al., 2016). They are macromolecules made up of repeating monomeric subunits linked by covalent bonds (Wen et al., 2018). Based on their origin, biopolymers are divided into natural and synthetic classes (Taghipour-Sabzevar et al., 2019). The advantages and disadvantages of these biopolymers are taken into consideration during selection for the development of a drug delivery system. Synthetic Biopolymers Synthetic biopolymers can be derived from natural polymers or chemically synthesized. They have attracted much attention because of their stability, flexibility, low immunogenicity, and biodegradability. Since they resist hydrolysis and can tolerate high temperatures, they can be heat-sterilized without degradation (Rahman and Hasan, 2019). Poly (a-hydroxy acids), polyhydroxyalkanoates (PHAs), poly (lactones), and poly(alkyl cyanoacrylates) (PACA) are the common synthetic biopolymers, among which poly (a-hydroxy acids) are the most employed class of biopolymers for production of PNs. Poly (a-hydroxy acids) are degraded by non-enzymatic hydrolysis of the ester linkage into non-toxic monomers (lactic acid and glycolic acid). Their degradation rate depends on intrinsic properties such as molecular weight, chemical structure and hydrophobicity (Doppalapudi et al., 2016). Nanoparticles made up of these polymers have been developed for the delivery of various hydrophilic and hydrophobic anti- cancer agents such as doxorubicin, 5-fluorouracil, cisplatin, paclitaxel, and docetaxel (Rafiei and Haddadi, 2017; Ashour et al., 2019; Domínguez-Ríos et al., 2019; Maksimenko et al., 2019; Mittal et al., 2019). PLG was the first polymer of this class investigated for biomedical application (Doppalapudi et al., 2016). It is synthesized through the polycondensation of glycolic acid or ring opening of glycolide, but it is not a good choice for the formulation of nanocarriers for cancer therapeutics because of its rigidity and rapid degradation (Shukla et al., 2019). PLA, another widely-investigated polymer, can be obtained from the polycondensation of lactic acid (LA) or by the ring opening polymerization of lactide; it exists in two isomeric forms, poly(L-lactic acid) and poly(D-lactic acid) (Fonseca et al., 2015). PLA naturally degrades in situ through the hydrolysis of the ester linkage, rendering LA and its short oligomers as the degradation products. Since the products of PLA biodegradation are cleared easily from the body, its use does not induce severe immune responses (Lee et al., 2016; Casalini et al., 2019). Among polyesters, PLGA is the most widely-used co-polymer for the development of targeted drug delivery systems, and is made up of glycolic acid and lactic acid monomers (Mir et al., 2017; Maity and Chakraborti, 2020). PLGA polymers undergo complete biodegradation in aqueous media and their characteristics can be altered by varying the chemical composition (lactide/glycolide ratio) and the chain length. For example, the degradation rate and the drug-release rate accelerate when the molecular weight of the copolymer is decreased (Molavi et al., 2020). PLGA can be prepared at different lactide/glycolide molar ratios such as 50/50, 65/35, 75/25, and 85/15. Lactide is more hydrophobic than glycolide, so a decrease in the proportion of lactide increases the rate of hydrolytic degradation of the copolymer, with consequent rapid release of the encapsulated drug (Gentile et al., 2014). It has been suggested that the degradation times of 50/50, 75/25 and 85/15 PLGA is 1-2, 4-5, and 5-6 months, respectively (Middleton and Tipton, 1998; Anju et al., 2020). Biopolymers produced by microorganisms have shown promise as a substitute for the synthetic polymers currently being used in the industry. For instance, PHAs are naturally produced and accumulated as energy/carbon storage material by many bacteria. PHAs have recently gained great attention because of their biocompatibility, biodegradability, thermoplasticity, low toxicity, and availability (Korde and Kandasubramanian, 2020). They are polyesters of various hydroxyalkanoate monomers that can be produced either through the natural bioconversion process or by chemical synthesis via the ring-opening polymerization of ẞ-lactones (Li and Loh, 2017). Poly(hydroxybutyrate) (PHB) is a PHA derivative used in targeted drug delivery due to its prolonged degradation time in vivo and its lesser effect on the pH of tissues as compared to the polylactides (Korde and Kandasubramanian, 2020). According to ISO 10993, PHB nanoparticles have been shown to be safe when used on animals (Masood, 2016). Among polylactone-based polymers, poly(Ɛ-caprolactone) (PCL) is the most studied polymer for anticancer drug development. It is a semicrystalline compound obtained by the ring-opening polymerization of ε-caprolactone (Witt et al., 2019). PCL exhibits slower ester bond hydrolysis at physiological pH and has a less acidic character than poly-hydroxy acids; in addition, the slower degradation rate of PCL prolongs the release of encapsulated drugs (Doppalapudi et al., 2016). Poly(alkyl cyanoacrylates) (PACA) are another biodegradable polymer class useful for developing nanocarriers. These polymers are mainly degraded through the hydrolysis of the ester bonds of their alkyl chain. The rate of degradation depends on the alkyl chain length: the longer the alkyl chain, the slower the rate. The two resulting products, namely alkyl alcohol and poly (cyano acrylic acid), are both soluble in water (Nicolas and Couvreur, 2009; Doppalapudi et al., 2016). PACAs can retain substantial amounts of drug (Sulheim et al., 2017). Poly(isohexylcyanoacrylate) nanoparticles containing doxorubicin (Livatag®-see section “Livatag®”.) have been proposed as an innovative formulation for human primary liver cancer and have reached phase III of clinical trials (Merle et al., 2017). Natural Biopolymers Natural biopolymers include animal- or plant-derived proteins and polysaccharides as well as polymers obtained from microbial sources. These are widely used in drug delivery research due to Frontiers in Pharmacology | www.frontiersin.org 5 February 2021 | Volume 12 | Article 601626See Answer
- Q3:TO DO: Summarize the following research paper and comment on the direction they could go next! (your own Idea). 250 words./n Review molecules MDPI Application of Conducting Polymer Nanostructures to Electrochemical Biosensors 1 Waleed A. El-Said ¹®, Muhammad Abdelshakour ¹, Jin-Ha Choi 2 and Jeong-Woo Choi 2,* 1 Department of Chemistry, Faculty of Science, Assiut University, Assiut 71516, Egypt; 2 * awaleedahmed@yahoo.com (W.A.E.-S.); muhammed.abdl_shakor@science.au.edu.eg (M.A.) Department of Chemical and Biomolecular Engineering, Sogang University, 35 Baekbeom-Ro, Mapo-Gu, Seoul 04107, Korea; jinhachoi@sogang.ac.kr Correspondence: jwchoi@sogang.ac.kr; Tel.: +82-2-705-8480 Academic Editor: Carlos Alemán check for updates Received: 24 December 2019; Accepted: 10 January 2020; Published: 12 January 2020 Abstract: Over the past few decades, nanostructured conducting polymers have received great attention in several application fields, including biosensors, microelectronics, polymer batteries, actuators, energy conversion, and biological applications due to their excellent conductivity, stability, and ease of preparation. In the bioengineering application field, the conducting polymers were reported as excellent matrixes for the functionalization of various biological molecules and thus enhanced their performances as biosensors. In addition, combinations of metals or metal oxides nanostructures with conducting polymers result in enhancing the stability and sensitivity as the biosensing platform. Therefore, several methods have been reported for developing homogeneous metal/metal oxide nanostructures thin layer on the conducting polymer surfaces. This review will introduce the fabrications of different conducting polymers nanostructures and their composites with different shapes. We will exhibit the different techniques that can be used to develop conducting polymers nanostructures and to investigate their chemical, physical and topographical effects. Among the various biosensors, we will focus on conducting polymer-integrated electrochemical biosensors for monitoring important biological targets such as DNA, proteins, peptides, and other biological biomarkers, in addition to their applications as cell-based chips. Furthermore, the fabrication and applications of the molecularly imprinted polymer-based biosensors will be addressed in this review. Keywords: conducting polymers; molecularly imprinted polymer; cell-based chip; nanotechnology; electrochemical sensor; biosensors 1. Introduction Conducting polymers (CPs) have emerged as one of the most promising materials in many biological and biomedical applications, including biosensors and tissue engineering applications [1–3]. The wide applications of the CPs are owing to their biocompatibility and their unique electrical properties that could convert the biochemical information into electrical signals. In addition, CPs have several functional groups, which provide maximum enzyme loading through the interaction between the enzyme molecules and the polymers' functional groups, thus a well-organized scaffold biosensors could be achieved [4]. Recently, nanostructured CPs represented an excellent building block for developing highly sensitive biosensors [5] due to their unique properties that combine with those of the CPs (biocompatibility, direct electrochemical synthesis) and the nanomaterials (e.g., large surface area, flexibility for the immobilization of biomolecules and quantum effect) [6–8]. Several synthetic strategies were reported for the nanostructured CPs (NSCPs) synthesis, including template-based (either hard or soft template) methods, template-free synthesis [9–12], as well as the physical approaches (e.g., electrospinning) [13]. Molecules 2020, 25, 307; doi:10.3390/molecules25020307 www.mdpi.com/journal/molecules Molecules 2020, 25, 307 2 of 11 Among the many NSCPs, polyaniline nanostructures (PANI NSs) have been mainly prepared with the aid of template-guided polymerization within channels of microporous zeolites, porous membranes, and chemical route in the presence of self-organized supramolecules or stabilizers [14–16]. PANI NSs have higher sensitivity and faster time response than its conventional bulk counterpart due to higher active surface area and shorter penetration depth for target molecules [16]. The high surface area and porous structure further allow the fast diffusion of molecules into the framework leading to their applicability as biosensors. The blend of metal nanoparticles with conjugated polymers to form nanocomposite is intended to increase electrical conductivity [17,18]. One of the most significant current discussions has clearly demonstrated that gold and silver nanoparticles could be embedded into a polymeric layer, which largely increased the surface area for modification of diverse biomolecules. In recent years, there has been considerable interest in the system of electrode modification using nanoparticles and conducting polymers [18–24]. 2. NSCP-Integrated Electrochemical Biosensors 2.1. Nscps for Electrochemical Detection of Glucose and H₂O2 fast Nowadays, diabetes mellitus represents a severe health problem worldwide due to its complications that are more harmful than diabetes itself [25]. Therefore, developing an accurate and assay for early diagnosis of diabetes disease is an urgently needed issue. Several analytical assays were reported for monitoring diabetes based on the measurement of glucose level in blood [26–28]. The glucose sensors could be classified into enzymatic and enzyme-free sensors [26–29]. Here, we will discuss the uses of CPs for developing highly sensitive glucose sensors. Deepshikha et al. report on the preparation of PANI NSs by using sodium dodecylsulphate (NSPANI-SDS) as glucose and H₂O2 biosensor [15]. SDS acts as an ideal structure-directing agent for the synthesis of ordered nanostructured polymer composed of framework protonated amine such as PANI NSs. The uses of these NSs polymer with large specific surface area could enhance the conductivity of PANI and results in easily immobilization with high loading of horseradish peroxidase (HRP) and glucose oxidase (GOx). In addition, these NSs enhance the rate of electron transfer and the current response. These modified PANI NSS were used as optical and electrical biosensors with good performances, fast response time, wide linear range, and good selectivity, stability and reproducibility. Furthermore, Abidian et al. have reported the fabrication and applications of PEDOT nanofibers for electrochemical detection of glucose based on entrapped of the glucose oxidase enzyme (GOx) into the PEDOT nanofibers during the galvanostatic polymerization process at Pt electrode [30]. This sensor has demonstrated a high sensitivity, high electrochemical stability and lower limit of detection (LOD) than the GOx-incorporated PEDOT film (PEDOT F-GOx) sensors (as shown in Figure 1a) that related to their large surface area. Soganci et al. have modified the graphite rod electrode with a super-structured CP composed of amine substituted thienyl-pyrrole derivative based on the electropolymerization process [31]. This CP is characterized by the presence of free amine groups that allowed the covalent bonding between the electrode and the biorecognition elements such as glucose oxidase (GOx). This sensor was applied for glucose detection in beverages. Munteanu et al. showed a dual electrochemical sensor with optical microscopy as an opto-electrochemical sensor for detecting both hydrogen peroxide and glucose [32]. The principal of the sensor is based on uses of osmium complex-based redox polymer hence its oxidation state could change during the interactions of hydrogen peroxide and glucose with the enzyme, which is a time-dependant interaction as depicted in Figure 1b,c. Molecules 2020, 25, 307 (a) (i) (iii) 2 mm 20 um (ii) 80 μm 1 um (b) Solution Solution Electrochemical Reduced oxidation Reduced ng hydrogel hydrogel ITO/FTO Ոչ Electrochemical reduction ITO/FTO Glass Glass n₁ Incident beam Reflected beam Incident beam Microscope objective no PEDOT GOX BFRLM image of reduced hydrogel Microscope objective BFRLM image of oxidased hydrogel (c) Electrochemical information with spatial resolution Current (HA) 0.2- 2222 Reflected beam E 0.130 V I=0.95x10 A 0.1- AE = 0.091 V 1/1-0.97 0.0- E=0.039 V 0.1- = 0.98x10" A -0μM H₂O₂ 200 KM HẠO, -0.2- 100 μm -0.2 0.0 0.2 0.4 Potential (V) Figure 1. (a) Glucose oxidase (GOx)-incorporated PEDOT on the microelectrode array: (i) Pt microelectrode array. (ii) Electrodeposition of GOx-incorporated PEDOT film (PEDOT F-GOx) with electrospinning of PEDOT nanofibers (PEDOT NFs-GOx), poly(L-lactide) (PLLA) nanofibers on the microelectrode array. (iii) Electrodeposition of PEDOT around the PLLA nanofibers to form GOx-incorporated PEDOT nanofibers (PEDOT NF-GOx). Optical and scanning electron microscope (SEM) images of the entire microelectrode array are below Reproduced with permission [30]. Copyright 2014 Wiley. (b) Schematics of BFRLM for increasing the spatial resolution of redox hydrogel-based electrochemical biosensors. The incident light is refracted and reflected on the different interfaces of the multilayered sensor [32]. (c) Electrochemical detection of hydrogen peroxide using an Fluorine doped Tin Oxide (FTO) electrode modified with horseradish peroxidase (HRP)-based redox hydrogel [32]. 2.2. Nscps for Cell-Based Chip Applications It is challenging to understand cell behavior based on the measurement of only nucleic acid or protein expression levels because the cells are much more complicated than the sum of its components [33]. Several electrically conductive scaffolds have been used for making a cell-based chip for enhancing the adhesion, proliferation, and differentiation of several cell types such as neurons [33-35]. Here, we will address the uses of CPs modified electrodes for developing cell-based chips and their applications. Lee et al. developed an electrochemical conducting scaffold composed of pyrrole N-hydroxyl succinimidyl ester and pyrrole (PPy-NSE) copolymer, then modified this copolymer with nerve growth factor (NGF) and it used for PC12 cells immobilizations [36]. They have claimed that cells have extended neurites similarly to that for cells cultured in medium containing NGF. El-Said et al. reported on uses of a thin layer of polyaniline emeraldine base (EB) coated indium-tin oxide (ITO) electrode as a cell-based chip [35]. On the contrary to the metal electrodes, PANI-EB/ITO electrode showed an excellent electrochemical activity at neutral pH without co-deposition of an acidic counterion. The developed electrode was used as a cell-based chip for quick and easy measuring cellular electrochemical properties, the cell viability, cell adhesion, cell proliferation and monitoring the effects of different anticancer drugs on the cell viability. The same group has reported the in-situ electrochemical synthesis of polypyrrole (PPy) nanowires with a nanoporous alumina template [37]. They have shown the formation of highly ordered porous alumina substrate and the growth of the PPy nanowires inside the nanoporous structures based on the direct electrochemical oxidation of the pyrrole monomer, as shown in Figure 2a. The cellular behavior, cell morphology, adhesion, and proliferation, as well as the biocompatibility of PPy nanowires/nanoporous alumina substrate towards both cancerous 3 of 11 Molecules 2020, 25, 307 4 of 11 and normal cells, were investigated compared with other substrates. They have demonstrated that the PPy nanowires/ nanoporous alumina substrate showed better cell adhesion and proliferation than other control substrates. This study showed the potential of the PPy nanowires/nanoporous alumina substrate as biocompatibility electroactive polymer substrate for both healthy and cancer cell cultures applications. Strover et al. have incorporated pyrrole and thiophene moieties in its monomer, to fabricated PolyPyThon (PPyThon)-based molecular brushes [38]. A film of this CP was deposited on the gold substrate used as a scaffold for electrical stimuli-responsive surfaces of human fibroblasts cells, as shown in Figure 2b. (a) Smooth Al foil 1st Anodization 1st anodized AI Anodization 2nd Anodization Pyrrole polymerization Cell culture Polypyrrole nanowires & Nanoporous alumina Cell culture Cell culture (b) Uncoated 2nd anodized Al Figure 2. (a) Schematic diagram represented the fabrication of different cell culture substrates and the cell immobilization process. Reproduced with permission [37]. Copyright 2010 Elsevier. (b) Human fibroblasts after two days in culture on PPyThon film on gold. Green stains Ki67 (proliferation marker), yellow stains vinculin (focal adhesion marker), red stains actin (cytoskeleton). Nuclei counterstained with DAPI (blue). The scale bar is 100 μm. Reproduced with permission [38]. Copyright 2013 Elsevier. Based on the above, uses of the cell-based chips is a promising alternative to animal experiments, due to the disadvantages of the uses of animal models because they are violating animals' rights, costly, time-consuming and also poor relevance to human biology. In addition, the uses of cell-based chips as biosensors for monitoring effects of anticancer drugs or for monitoring the differentiation of the neural cells showed many advantages that including (1) increasingly more sophisticated representation of absorption, distribution, metabolism, excretion, and toxicity (ADMET) process, (2) better understand the drug interaction mechanisms in the human body, and (3) showed a great potential to better predict drug efficacy and safety. 2.3. Nscps for Different Biosensor Applications Conducting polymers have been widely used for preparing of sensor platforms and imparts many advantages due to the incorporation of their functional groups into their fabrication. Here, we will discuss the fabrication and uses of different NSCPs and their composites, as well as their electrochemical biosensor applications for the various biological targets such as nucleic acid, ATP, neurotransmitter, etc. Guanine (G) and adenine (A) and are two of the purine bases, which participate in the building of nucleic acids and are fundamental compounds in different biological systems. The abnormal concentration of A and G in body fluid is related to the deficiency of the immunity system. Hence, monitoring of the A and G concentration in living organisms is a great demand issue. El-Said et al. have fabricated poly(4-aminothiophenol) (PATP) nanostructures layered on gold nanodots patterned indium tin oxide (ITO) electrode based on a simple method as shown in Figure 3a [39]. The modified gold nanodots ITO electrode was fabricated based on thermal evaporation of pure Au metal onto the ITO surface through polystyrene monolayer. Then, use of these Au nanodots as a template for self-assembly immobilization of ATP molecules followed by electrochemical polymerization of ATP into PATP. The modified electrode was applied to monitor the concentration of the mixture of adenine Molecules 2020, 25, 307 5 of 11 and guanine with LOD of 500 and 250 nM, respectively. Furthermore, the modified electrode was extensively applied for detecting adenine and guanine in human serum. Aksoy et al. have developed a selective electrochemical dopamine biosensor based on polyimide (PI) and polyimide-boron nitride (PI-BN) composites as a selective membrane for dopamine detection [40]. The introduction of BN particles into the PI matrix results in enhancement of the sensitivity, selectivity, and reversibility (i.e., the rapid electron transfer), with a LOD of about 4 × 10-8 M. Hybridization of PANI with nanomaterials could endow great promise in the sensors field due to the enhancement of its electrical conductivity in addition to its capability to act as a scaffold for immobilization of the biological species [41]. Pseudomonas aeruginosa (P. aeruginosa) is among the most common pathogenic gram-negative bacteria that could cause corneal ulcers and blindness within two days. Pyocyanin (PYO) is the biomarker that has been used for monitoring P. aeruginosa. Elkhawaga et al. have prepared PANI/Au NPs/ITO electrode as PYO sensor in a culture of P. aeruginosa. The results indicated that PANI/Au NPs/ ITO electrode is more sensitive toward PYO biomarker than either bare ITO electrode or Au NPs/ITO electrode with LOD of 500 nM [42]. The enhancement of the electrochemical activity of PANI/Au NPs/ITO modified electrode towards the PYO related to the presence of positive charges on its surface that could enhance the mass transfer rate of the negatively charged PYO based on the electrostatic attraction force. The same group has extended the uses of PANI/Au NPs/ ITO electrode for diagnosis of P. aeruginosa in 50 samples collected from patients suffering from corneal ulcers as shown in Figure 3b [43]. The obtained results were compared with the results gained by the screen-printed electrode (SPE), conventional techniques, automated identification method, and the amplification of the 16 s rRNA gene by polymerase chain reaction (PCR) as a standard test for P. aeruginosa identification. The electrochemical detection of PYO by square wave voltammetry (SWV) technique using PANI/Au NPs modified ITO electrode was the only technique showing 100% agreement with the molecular method in sensitivity, specificity, positive and negative predictive values when compared with the SPE, conventional (including colony morphology, pigment production, and biochemical tests) and automated (including the automated ID and Ast system and the PCR) methods. Thus, PANI/Au NPs/ITO electrode is recommended as a fast, cheap, accurate, and selective PYO biomarker sensor in P. aeruginosa in the corneal ulcer cases based on the SWV technique. Zika virus (ZIKV) is a flavivirus. Recently, there is an increasing interest in developing a rapid Zika virus identification assay due to the appearance of the viral infection in infants. Tancharoen et al. report the development of a new type of ZIKV electrochemical biosensor [44]. This biosensor consisted of surface imprinted polymers (SIPs)/graphene oxide composites, as shown in Figure 3c. The LOD of this sensor agreed with that of the RT-PCR method, in addition to the capability of this sensor to detect the Zika virus in the presence of the dengue virus and serum samples. Lactate is one of the cellular metabolites, which is associated with some critical health care conditions. Pappa et al. reported the fabrication of a micrometer-scale polymer-based transistor platform for the detection of lactate, as shown in Figure 3d [45]. The uses of the electron-transporting (n-type) organic polymer incorporate hydrophilic side chains that have enhanced the ion transport/injection, facilitated the enzyme conjugation and acted as a series of redox centers. The developed sensor showed a fast, selective, and sensitive metabolite capability.See Answer
- Q4: Review prosthesis MDPI Functionalization of Polymers and Nanomaterials for Biomedical Applications: Antimicrobial Platforms and Drug Carriers Masoud Delfi 1,+, Matineh Ghomi 2,+, Ali Zarrabi 3,4,+ Zahra Baghban Taraghdari 6,+, Milad Ashrafizadeh 7 Tarun Agarwal 9 5,t, Reza Mohammadinejad Ehsan Nazarzadeh Zare 8,*, Vinod V. T. Padil 10,* D, Babak Mokhtari 2, Filippo Rossi 11 Giuseppe Perale 12,13, Mika Sillanpaa 14,15,16,17,Assunta Borzacchiello 18, Tapas Kumar Maiti 9,* and Pooyan Makvandi 2,18,* D 1 2 3 4 5 6 7 8 9 Department of Chemical Sciences, University of Naples “Federico II", Complesso Universitario Monte S. Angelo, Via Cintia, 80126 Naples, Italy; Masoud.delfi@unina.it Chemistry Department, Faculty of Science, Shahid Chamran University of Ahvaz, Ahvaz 6153753843, Iran; ma_gh@rocketmail.com (M.G.); bmokhtari4@gmail.com (B.M.) Sabanci University Nanotechnology Research and Application Center (SUNUM), Tuzla 34956, Istanbul, Turkey; alizarrabi@sabanciuniv.edu Center of Excellence for Functional Surfaces and Interfaces (EFSUN), Faculty of Engineering and Natural Sciences, Sabanci University, Tuzla 34956, Istanbul, Turkey Pharmaceutics Research Center, Institute of Neuropharmacology, Kerman University of Medical Sciences, Kerman 76169-13555, Iran; r.mohammadinejad87@gmail.com Department of Chemical, Materials & Industrial Production Engineering, University of Naples Federico II, 80125 Naples, Italy; z.baghban@studenti.unina.it Department of Basic Science, Faculty of Veterinary Medicine, University of Tabriz, Tabriz 51666-16471, Iran; dvm.milad1994@gmail.com School of Chemistry, Damghan University, Damghan 36716-41167, Iran Department of Biotechnology, Indian Institute of Technology, Kharagpur 721302, India; tarun3agarwal5@gmail.com 10 Department of Nanomaterials in Natural Sciences, Institute for Nanomaterials, Advanced Technologies and Innovation (CXI), Technical University of Liberec (TUL), Studentská 1402/2, 46117 Liberec, Czech Republic Department of Chemistry, Materials and Chemical Engineering, Politecnico di Milano Technical University, 20133 Milano, Italy; filippo.rossi@polimi.it 11 12 13 14 15 56 Faculty of Biomedical Sciences, University of Southern Switzerland (USI), Via G. Buffi 13, 6900 Lugano, Switzerland; giuseppe.perale@supsi.ch Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Donaueschingenstrasse 13, 1200 Vienna, Austria Institute of Research and Development, Duy Tan University, Da Nang 550000, Vietnam; mikaetapiosillanpaa@duytan.edu.vn Faculty of Environment and Chemical Engineering, Duy Tan University, Da Nang 550000, Vietnam 16 School of Civil Engineering and Surveying, Faculty of Health, Engineering and Sciences, 17 University of Southern Queensland, West Street, Toowoomba, QLD 4350, Australia Department of Chemical Engineering, School of Mining, Metallurgy and Chemical Engineering, University of Johannesburg, P. O. Box 17011, Doornfontein 2028, South Africa 18 Institute for Polymers, Composites, and Biomaterials (IPCB), National Research Council (CNR), 80125 Naples, Italy; bassunta@unina.it * Correspondence: ehsan.nazarzadehzare@gmail.com (E.N.Z.); vinod.padil@tul.cz (V.V.T.P.); maititapask@gmail.com (T.K.M.); pooyan.makvandi@ipcb.cnr.it or Pooyanmakvandi@gmail.com (P.M.) + Equaul co-first author. Received: 21 May 2020; Accepted: 19 June 2020; Published: 23 June 2020 check for updates Abstract: The use of polymers and nanomaterials has vastly grown for industrial and biomedical sectors during last years. Before any designation or selection of polymers and their nanocomposites, Prosthesis 2020, 2, 117–139; doi:10.3390/prosthesis2020012 www.mdpi.com/journal/prosthesis Prosthesis 2020, 2 118 it is vital to recognize the targeted applications which require these platforms to be modified. Surface functionalization to introduce the desired type and quantity of reactive functional groups to target a cell or tissue in human body is a pivotal approach to improve the physicochemical and biological properties of these materials. Herein, advances in the functionalized polymer and nanomaterials surfaces are highlighted along with their applications in biomedical fields, e.g., antimicrobial therapy and drug delivery. Keywords: drug delivery; surface functionalization; antibacterial activity; antimicrobial properties; polymeric nanoparticles 1. Introduction Polymers are the most used compounds which possess many advantages, such as lightweight materials, cheap, easy to produce as different products, and long durability [1]. To improve the applicability and specificity of polymers, some modifications to the particles are needed. These changes can be made in the bulk of the carrier, like incorporating Cu in nanospheres to enhance imaging and photocatalytic properties [2]. Another major site of modification is the surfaces of the polymers. Dry surface treatment techniques e.g., corona discharges [3], oxygen plasma [4], ultraviolet light-ozone [5], and cold atmospheric jet [6] have been applied for improving polymer/polymer or polymer/ceramic adhesions. To make corrections in the polymer surfaces, wettability properties of polymers can be altered to govern the adhesion of various coatings. This means the ability of the liquid for moistening the surface of a polymer, which in turn, creates different coatings on the surface. It is worth noting that polymers often have very poor wetting properties that prevent the coatings from sticking properly [7]. In this regard, surface tension plays a critical role on the surface wettability of the polymers, while low surface tension creates less wetting tendency. The attractive forces that exist on the microscopic scale of polymer surface molecules or atoms, prevent them from leaving the surface of solid/liquid materials. These forces are dependent on the binding energy of the atoms/molecules in the solid/liquid materials and cause the surface energy to disperse. For catching the high wettability on the polymer surfaces, a high concentration of polar components should be induced [8]. For improving the various properties (e.g., thermal, mechanical, and optical) of polymers, they can be combined with nanoparticles and nanofillers to form nanocomposites. Nanoparticles can modify the wetting tendency of polymer surfaces through two mechanisms. (I) Alterations of the intermolecular interactions of interfacial solid-water and enhancing the wettability of the polymer surface through modifying the chemical composition, (II) addition of some nanoparticles that can increase the hydrophobicity or water-repellence of the polymers via modification of the surface morphology (e.g., surface roughness) [9]. The physical and chemical properties of the polymer surfaces can be modified by the mentioned methodology for intimate interfacial contacts between two different phases, while these modifications have no effect on the properties of the bulk. These surface treatments show significant efficiencies such as (I) alteration of the chemical structure of polymer surfaces by free-radical reactions of the polymer surface with the surrounding gases, (II) etching of a microscopic layer or selective leaching of polymeric chains with low molecular weight, (III) polymer reinforcement by increasing the cross-links or branching of the polymer molecules, and (IV) cleaning by converting liquid or solid films/contaminants into volatile gas products [10,11]. Prosthesis 2020, 2 119 The purpose of this review is to introduce the advances in surfaces functionalization of polymer and nanomaterials and discuss their recent investigations toward antibacterial modification and drug delivery (Figure 1). Polymer chain Antimicrobial agent Antimicrobial activity DOXX Functional group for targeted delivery Drug Delivery Drug encapsulated in nanoparticle Figure 1. Surfaces functionalization of polymer and nanomaterials for antimicrobial therapy and drug delivery applications. 2. Antimicrobial Therapy Humans are always exposed to the threat of microbial infections regardless of place and time [12,13]. There are three ways to impart antimicrobial activity to a platform (Figure 2). The first is functionalization of polymers or nanomaterials with antimicrobial agents, e.g., quaternary ammonium compounds (QACs) [14,15]. These compounds have been extensively used to improve the antimicrobial efficacy of various surfaces through a contact-killing mechanism. QACs, in particular the ones possessing long alkyl chains, are mostly utilized as antimicrobial and disinfectant materials. These compounds exhibit strong toxicity against fungi and amoebas, and have the ability to envelop viruses as well [15,16]. Another example that can be mentioned in this context is the surface modification of polysaccharides using QACs. Due to the available functional groups of polysaccharides, they are known as abundant renewable bio-substrates. For instance, glycidyl trimethyl ammonium chloride grafted cellulose and chitosan films have been investigated extensively as antibacterial surfaces [17-19]. Prosthesis 2020, 2 Alive microbes 3 2 Using antimicrobial platforms Dead microbes Antimicrobial agent Antimicrobial drug Surface functionalized nanoparticle/polymer Drug delivery systems ww Antimicrobial nanoparticle Polymer chain Nanocomposite 120 Figure 2. Schematic illustration of three ways of antimicrobial activity in a platform: (left panel) surface functionalization of nanoparticles/polymers, (middle panel) drug delivery system, and (right panel) fabrication of nanocomposite. The second approach is fabrication composites by adding fillers such as metal-based nanomaterials like Ag nanoparticles [20–22]. Silver has been used for wound healing soon after its discovery as an effective antimicrobial agent. It can be used as a solid state (powder) or even salt solutions for wound treatment [23,24]. Although it has been used for a very long time, the exact mechanism of action of silver nanoparticles remained partially unknown. The antimicrobial activities of Ag NPs can be divided into four steps: approaching to the bacterial surface, disruption of the cell wall of the bacteria and its membrane through changing its permeability, exerting toxicity effects and oxidative stress by producing ROS and free radicals, and modulation of signal transduction pathways [25]. The last method is encapsulation of antimicrobial drugs or biomolecules such as gentamicin. The drug is a potent broad-spectrum antibiotic with high toxicity efficacy against various Gram-positive and Gram-negative organisms [26]. Regarding the killing strategy, there are two main strategies to confront bacteria: (I) releasing of antibacterial agents on the infected sites and (II) attaching to surfaces containing antibacterial compounds (contact-killing). In the latter strategy, surface modification of metallic or polymeric surfaces is performed to attach various agents with antibacterial activities to make direct contacts with microorganisms. To increase the antimicrobial efficacy, implant functionalization using antimicrobial peptides (AMPs) has been introduced to be a very practical technique. An interesting example in this respect, a polydopamine coated Ti substrate was conjugated with AMPs through a click chemistry reaction (Figure 3). The AMPs show resistance against the infection of different types of microbes including bacteria and fungi. They can penetrate the bacterial cell membrane and damage the drug-resistant bacteria vigorously. The surfaces functionalized by AMP showed high microbicidal effects with low cytotoxicity [27]. Prosthesis 2020, 2 (b) AMP: KRWWKWWRR Surface a HO Dopamine Dopamine: HO OH OH 2-Bromoisobutyryl bromide: Ti-PDA Br Surface b 2-Bromoisobutyryl bromide Br Br Ti-Br Surface c NH NIPAM Ti-pNIPAM Surface f NH NaN Br N3 N3 Ti-Na Surface d NaN Ti-PNIPAM-N₁ Surface g Substrate glass, polymer, metal SEBS bead SEBS chain NH H₂N PraAMP: KRWWKWWRR HO PDA layer: NH2 HO PraAMP PraAMP NIPAM: H₂N H₂N Ti-AMP Surface e HN- H₂N H₂N Ti-PNIPAM-AMP Surface h SEBS/p-xylene/decanol (polymer/solvent/nonsolvent) 346 Dry decanol (nonsolvent) Solvent evaporation NH p-xylene (solvent) 121 Figure 3. (a) Schematic of preparation of antibacterial implants through conjugation of antimicrobial peptides (AMPs) on Ti substrate. Reprinted with permission from [27]. (b) Schematic of preparation of biocompatible and antibacterial surfaces by superhydrophobic coating of styrene-b-(ethyleneco- butylene)-b-styrene elastomer (SEBS) on different substrates. Reprinted with permission from [28]. There are some ceramic nanocompounds, e.g., SiO2 nanoparticles, that do not possess antimicrobial activity yet have been modified to generate microbicidal materials. As an example, Makvandi et al. have functionalized silica nanoparticles with quaternary ammonium methacrylate blended withSee Answer
- Q5: PHA223 Rapid Review 2023-2024 Guidance Notes & Generic Marking Scheme Topic:-Development of Nanomedicine Strategies to Target Coronavirus Article link :- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457919/ Requirements - 3000 words (without reference) - you can use a maximum of 6 references - make sure that u including every single point - highlighting the small topic that in each section Structure of your assignment The required length of this assignment is 3000 words (+/- 10%), excluding references. A penalty will be applied if the word count falls below or exceeds this limit in accordance with standard university procedures. No special adjustments on words counts will be made for figures, tables, or flow charts. The word count will be adjudicated using Microsoft Word. Guidance on Assignment Structure 1. INTRODUCTION You must outline your question and the aims of your literature search. This section needs to set the scene of your topic by including relevant background information surrounding key concepts of your topic. In your introduction, you will give definitions and provide sufficient information for the reader to understand your topic and what you will be searching for in the literature. Your introduction should be structured in a way so that a reader understands exactly what your literature search will focus on. 2. METHODOLOGY In this section, you need to clearly explain your search strategy. Everything you do to identify selected articles for discussion must be clearly stated in this section. This includes outlining which key words were used, which databases were searched, when the databases were searched and any search criteria/filters that were applied. If a reader was to screen your methodology, they should arrive at the same articles as you did if you outlined your processes effectively. You must also include details of your inclusion/exclusion criteria that you apply when screening each article. You must also include a PRISMA flow diagram outlining your process of selecting articles and at what stages you applied your inclusion/exclusion criteria. To support you with your methodology and identification of suitable articles, please refer to the Rapid Review lecture and the online recording provided by the library services team. 3. RESULTS & CRITICAL APPRAISAL You must present your literature findings to the reader and therefore, the evidence for the proposition or therapeutic intervention you have outlined in your topic. This must include an effective critique of the identified literature with associated interpretation and link back to your topic. You will be expected to analyse your selected articles for the strengths and weaknesses of their conclusions based upon the quality and appropriateness of their studies. It will be beneficial to create a “summary table" of your literature findings which outlines some key information relating to each article included. 4. DISCUSSION & CONCLUSION You will need to finish your report by summarizing your findings and explaining relevance into practice/science. You will need to make clear concluding statements as to whether your literature findings have a clear outcome and ensure that your conclusion directly relates back to your topic. 5. REFERENCES Full in text citations are essential in addition to a full, accompanying bibliography. Either Harvard or Vancouver referencing style will be permitted. This will not form aprt of your word count. Oral Viva In addition addition to the written work you will submit, you will be asked to present yourself for an oral discussion with your tutor, probably after the Easter break. The conversation will be related to the dissertation that you have submitted and can be expected to last approximately 20 minutes. The exact dates and times that you must attend this meeting will be announced later in the term. The assignment comprises 30% of the available marks for PHA223. Within this 30%, 90% of the marks are attributed to the written work and 10% of the available marks are available via the oral defence. As the member of staff you will be holding your discussion with will want to read your written report before talking to you, it is imperative that you hand in your work by the deadline as stated above. Failure to hand in your work on time will therefore also have a negative impact on your ability to perform well in the oral discussion component of this assignment as well as the written component. Any problems or questions, please contact Alex To support you in the development of your review, I have created a very brief checklist of the key components I expect your review to contain (please see next page). 3 Appendix 1 – PRISMA Guides - he following link will support you in writing your report. This is a direct link to the PRISMA diagram which must be included within your methodology section. PRISMA 2020 flow diagram new SRS v1.docx (live.com) The following link directs you to the PRISMA checklist. This will support you in developing your own reviews. A checklist for use in your report has also been produced for you which links to the PRIMSA checklist. Microsoft Word - PRISMA 2009 Checklist.doc (prisma-statement.org) Biology of the virus Surface proteine Structure of proteine SRNA,DNA, transmetion (incoded genes) why we don't use the proteine it self in the body? Bread about the vaccien, applications ☹important of nanomedicien esearch for other therapates drugs for (could) example => clorophein - not very effectiev) Eviruse behaffier inside the body (particelse) ∞other uses of nanomedicen Res peratory system Title Spike Serves protein Included Appendix 2 - Checklist Section Introduction Background Aim Methodology Search terms and database Eligibility Criteria Search Strategy Data Collection Article Information Bias Assessment Synthesis methods Study selection Results & Discussion Summary table Synthesis of results Summary of findings Conclusion Conclusion Sumerias Checklist item Title clearly stated. Outline definitions and key concepts relating to your topic. State the aim of the review (chart) Draw Specify all databases, search engines (and all other sources) used to identify your articles. Date ranges of searches should be included. Clearly state the inclusion and exclusion criteria used to select your articles State all filters applied during your literature searches State how you decided whether a study met inclusion criteria and how you screened each article to determine this. Provide details of outcomes for which data was sought (for example: article types, participant characteristics, intended outcomes) Provide details on how you reduced risk of bias within your own searches Outline how you will document your findings Include the PRISMA flow diagram Brief summary of each article identified for inclusion (optional) Critically appraise the findings from each article and integrate findings into core themes. Ensure that all interpretations are linked and discussed in relation to your research topic. Strengths and weaknesses of the articles should be highlighted and discussed to provide context into your discussion. Assessment of bias within your articles should also be included. Where necessary, discussions of statistical significance should also be made. Discuss the findings in relation to what is already known about your topic. Do they support what is already known, do they add new information, or do they disagree with previously outlined facts? General overview of findings and implications linked to your topic. Ensure this is clear, concise, and not a repetition of previously discussed findings. Outline whether any future research is required as a result of your findings or if there is a clear conclusion from your gathered information. References References Full list of bibliography references linked to in text citations. Use Harvard or Vancouver referencing style Draft 22/2 Ginars onmeron marchSee Answer
- Q6: ELSEVIER Materials and Design 201 (2021) 109484 Contents lists available at ScienceDirect Materials and Design journal homepage: www.elsevier.com/locate/matdes Physical and biological engineering of polymer scaffolds to potentiate repair of spinal cord injury a,1 Yiqian Luo ª‚¹, Fei Xue 1, Kai Liud, Baoqin Li ª, Changfeng Fu ª,*, Jianxun Dingb a Department of Spine Surgery, The First Hospital of Jilin University, 1 Xinmin Street, Changchun 130021, PR China b Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, 5625 Renmin Street, Changchun 130022, PR China c School of Chemistry and Life Science, Advanced Institute of Materials Science, Changchun University of Technology, 2055 Yan'an Street, Changchun 130021, PR China Department of Hand and Foot Surgery, The First Hospital of Jilin University, 1 Xinmin Street, Changchun 130021, PR China d Check for updates GRAPHICAL ABSTRACT • HIGHLIGHTS Polymer scaffolds possess enormous po- tential in the repair of spinal cord injury. • The optimized physical properties of scaffolds provide a favorable microenvi- ronment for spinal cord injury repair. • The biologically functionalized scaffolds reverse the adverse factors during spinal cord injury repair. FN Physical support Mechanical performance Internal structure Biodegradation Conductivity Bioelectrical signal conduction ✓ Physical functions - Polymer scaffold Biological functions- Endogenous neurogenesis NSC differentiation Axon growth Antioxidant M1| Macrophage polarization M2 MO Microenvironment regulation materials DESIGN ARTICLE INFO Article history: Received 19 November 2020 Received in revised form 2 January 2021 Accepted 12 January 2021 Available online 16 January 2021 Keywords: Polymer scaffold Spinal cord injury repair Physical support Signal transduction Microenvironment regulation Endogenous neurogenesis ABSTRACT Although the mortality rates of patients suffering from spinal cord injury (SCI) have decreased as the modalities of clinical therapy have been improved, the recovery of motor and sensory functions remains a challenge, ulti- mately leading to paraplegia or quadriplegia. Recently, neural tissue engineering scaffolds with appropriate phys- ical and biological functions have been extensively developed to promote nerve regeneration and improve motor and sensory functions during SCI therapy. In this work, we summarized the physical support and bioelectrical sig- nal conduction of polymer scaffolds for SCI repair from the aspects of biocompatibility, biodegradation, internal structure, mechanical performance, and conductivity. In addition, the biological functions of the polymer scaf- folds were reviewed for the reversal of adverse pathophysiological factors to improve the microenvironments of the injured site and promote endogenous neurogenesis during SCI therapy. Moreover, the future development of these engineered scaffolds for potential clinical applications was predicted. © 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Contents 1. Introduction. 1.1. Overview of spinal cord injury * Corresponding author. E-mail address: fucf@jlu.edu.cn (C. Fu). 1 Y. Luo and F. Xue contributed equally to this work. https://doi.org/10.1016/j.matdes.2021.109484 0264-1275/Ⓒ 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2 2 materialstoday Y. Luo, F. Xue, K. Liu et al. 1.2. 1.3. 1.4. 2. 3. 4. 5. Microstructure of spinal cord. Pathophysiology of spinal cord injury Treatment strategies for spinal cord injury. Physical support of scaffolds for spinal cord injury repair. 2.1. Biocompatibility and biodegradability. 2.2. Internal structure. 2.3. Mechanical performance. Bioelectrical signal conduction of scaffolds for spinal cord injury repair Microenvironment regulation by scaffolds for spinal cord injury repair 4.1. Antioxidants 4.2. Regulation of macrophages. Endogenous neurogenesis induced by scaffolds for spinal cord injury repair 5.1. Promoting axon growth 5.2. Tuning endogenous neural stem cells 6. Concluding remarks and future perspectives Declaration of Competing Interest Acknowledgments References. Materials and Design 201 (2021) 109484 2 2 3 4 4 4 6 6 8 8 10 10 11 13 13 15 15 15 1. Introduction 1.1. Overview of spinal cord injury Spinal cord injury (SCI) can interrupt the connection between the brain and peripheral organs, leading to dysfunction, such as paraplegia or quadriplegia [1]. There are various causes of SCI, including traffic ac- cidents, industrial accidents, sports injuries, and so forth. Particularly, traffic accidents account for 45.4% of all SCI cases, falling accidents for 27.4%, heavy objects falling for 10.8%, and tumble for 6.5% [2]. More than 20 million patients are currently suffering from SCI worldwide, with an increase of approximately 700,000 people per year [3]. Such a difficult situation has brought considerable burden to the society, and the clinical therapeutics do not achieve satisfactory results. Currently, neural tissue engineering strategies are the most promising modality among the numerous treatment approaches being explored. In this review, we described the microstructure of the spinal cord, pathophysiology of SCI, clinical therapy strategies, and advantages of neural tissue engineering. After that, we summarized the biocompatibil- ity and biodegradability of polymer scaffolds and highlighted the signif- icance of internal structure, mechanical performance, and conductivity for SCI repair. In addition, we reviewed the past research results to show the development and advantages of neural tissue engineering scaffold-mediated SCI therapy through microenvironment regulation and promotion of endogenous neurogenesis (Fig. 1 and Table 1). This work provided a comprehensive review of the theoretical basis and ap- plication of neural tissue engineering scaffolds for SCI repair. 1.2. Microstructure of spinal cord The spinal cord is an essential part of the central nervous system. Its primary function is to conduct nerve signal transduction and process low-level non-conditioned reflexes [4]. In particular, the anatomical mi- crostructure of the spinal cord provides the basis for the orderly mainte- nance of physiological functions. From the cross-sectional view of the spinal cord, the center of the spinal cord is the longitudinal central canal that connects with the cerebral ventricle. The butterfly-shaped gray matter surrounds central canal, and the outer layer of the gray mat- ter is the white matter [5,6]. A small amount of cerebrospinal fluid in the central canal protects and supplies nutrients to the brain and spinal cord. The gray matter is composed of neurons, glial cells, and blood vessels. The aggregation of neurons forms a complex neural circuit called the processing center for low-level non-conditioned reflexes, such as myotatic, flexion, uri- nation, and defecation reflexes [7,8]. The white matter is composed of nerve fibers and fibrous astrocytes. Nerve fibers are longitudinally arranged into bundles, which are divided into ascending conduction bundles and descending conduction bundles. They transmit signals between the brain and peripheral organs. In brief, ascending conduc- tion bundles transmit the information that controls the sensation of the body, whereas descending conduction bundles mainly transmit motor information from the brain to skeletal muscles of the trunk and limbs [9,10]. 1.3. Pathophysiology of spinal cord injury The pathophysiological process of traumatic SCI can be classified into mechanical (primary) and secondary injuries [11,12]. Mechanical damages directly destroy specific structures of the spinal cord, such as gray matter, vascular system, blood-spinal cord barrier, and nerve con- duction bundles [13]. During a secondary injury, necrotic debris of the myelin sheath, aggregation of various active inflammatory cells, such Physical functions Mechanical Physical support Internal structure 2 performance Biodegradation FN NSC differentiation Endogenous neurogenesis Biological functions Axon growth M1 Polymer scaffold Conductivity Signal transduction MO M2 Antioxidant acrophage arization Microenvironment regulation Fig. 1. Physical and biological functionalities of polymer scaffolds for promotion of SCI repair. Y. Luo, F. Xue, K. Liu et al. Table 1 Preparations and functions of polymer scaffolds. Materials and Design 201 (2021) 109484 Material Carrying substance HA CNTF HA/MA NPCs CS/MA IFN-y and NSCs CS/Col Col CBD-PlexinB1-LBD Agarose Matrigel CBD-EphA4-LBD and Template method PEG Gel/PLLA Gel/MA NSLCs iNSCs PCL SCs and iNSCs PLLA/Poloxamer CAB HEMA/MA Heparin/Col PEDOT/Agarose РРУ/ТА NPCs and bFGF Electrospinning Chemical crosslinking Free radical cryopolymerization Electrodeposition Chemical crosslinking Fabrication technology Biological function Reference Mesh filtration Activate NSCs, and facilitate their migration to the lesion area and differentiation into mature neurons [37] Photocrosslinking Promot the differentiation of NPC into neurons [38] Click chemistry and UV crosslinking 3D printing Neurofilament fibers extend from the host tissue to the scaffold Decrease the formation of scar and cavity, and improve the regeneration of nerve fibers [39] [41] Chemical crosslinking Facilitate axonal regrowth and remyelination of the regenerated tissue [43] Enhance linearly organized axon regeneration, and guide the reconnection of [45] functional axons Water/oil emulsion method, free radical polymerization Inhibit the growth of glial scars, and provide an aligned structure to direct axonal regeneration [46] Co-electrospun Photocrosslinking Support the proliferation and differentiation of NSLSC into motor neurons Decrease inflammation and cavity formation, facilitate axonal and neuronal regeneration, and inhibit glial scar hyperplasia [47] [48] Increase tissue remodeling, and secrete neurotrophic factor Reduce fibrotic scarring, and boost nerve regeneration [52] [53] Promote the orientational regeneration of neurons, and bridge the spinal cord [55] stumps in the conduit Promote axonal growth [56] PANI/CS/Gel PCL/Melatonin OE-MSCs SCs Activate endogenous NSCs neurogenesis in the lesion area Promote the differentiation of OE-MSCs into motor neurons [57] [60] 3D multilayer molding Reduce post-traumatic inflammation and oxidative stress, and regulate impaired mitochondrial function [120] HA/MnO2 NP MSCs Improve the stem cell adhesive growth and nerve tissue bridging, and alleviate the oxidative microenvironments [121] Silk fibroin AECs Inhibit local inflammation, promote myelin regeneration, and enhance nerve fiber regeneration [123] PHEMA Melt casting Promote the polarization of macrophages to M2 phenotype, reduce fibrosis, and promote angiogenesis [129] PCL IL-10 Electrospinning Induce macrophage polarization toward the M2 activated state [132] PLGA Oil-in-water single emulsion solvent Induce macrophages to polarize to M2 phenotype, reduce scar formation, and [136] Fibrin HA evaporation Electrospinning and concurrent molecular self-assembling Enzyme crosslinking CS NT-3 and NSCs promote axonal regeneration Guide axon regeneration directionally, and promote directional invasion of host cells and reconstruction of vascular system [142] Promote axonal regeneration and protruding connection Support the growth and reproduction of NSCs, and promote the differentiation [162] of NSCs into neurons [144] Abbreviations: 3D, three-dimensional; AECs, amniotic epithelial cells; bFGF, basic fibroblast growth factor; CNTF, ciliary neurotrophic factor; Col, collagen; CS, chitosan; Gel, gelatin; HA, hyaluronic acid; IFN-y, interferon-y; IL-10, interleukin-10; iNSCs, induced pluripotent stem cells-derived neural stem cells; MA, methacrylic anhydride; MP, methylprednisolone; NPCs, neural precursor cells; NSCs, neural stem cells; NSLCs, neural stem-like cells; NT-3, neurotrophin-3; OE-MSCs, olfactory ecto-mesenchymal stem cells; PCL, poly(&-caprolactone); PEG, poly(ethylene glycol); PEGDA, poly(ethylene glycol) diacrylate; PHEMA, poly(2-hydroxyethylmethacrylate); PLGA, poly(lactic-co-glycolic acid); PLLA, poly(L-lactic acid); SCs, Schwann cells; TA, tannic acid; UV, ultraviolet. as granulocytes and macrophages, and active glial cell populations can be observed in the harsh local microenvironments [14]. According to the time after injury and pathological mechanism, the secondary injury can be divided into three stages: acute stage, subacute stage, and chronic stage [15]. The acute phase is defined as the 48 h period after SCI. The main pathological changes were bleeding due to vascular injury, resulting in tissue edema and ischemia. With the de- struction of microcirculation, the subsequent pathological changes, such as ion imbalance, excitatory toxins, excessive production of free radicals, and inflammatory reactions, lead to further damage of neurons and glial cells. The subacute phase is from 48 h to two weeks after in- jury. The main pathological processes include phagocytosis of the in- flammatory corpuscle and reactive proliferation of astrocytes [16]. In particular, phagocytosis is the characteristic of this stage. The reactive proliferation of astrocytes leads to glial scarring, which is a crucial barrier to axonal regeneration [17]. The definition of chronic stage is generally considered to be six months or more after SCI. The main path- ological features of chronic stage include the formation and develop- ment of scars [18,19]. The use of various therapy measures, such as regulating physical properties and endowing biological functions, to en- hance the regeneration of damaged axons and myelin sheath, is the pur- pose of treatment at this stage, while preventing the formation of scars is the current promising research direction. 1.4. Treatment strategies for spinal cord injury Clinically, SCI is usually accompanied by destruction of the vertebral column. Therefore, surgical therapy is an essential modality for SCI treatment. Surgical operations can reestablish the stability of the spine to relieve compression of the spinal cord, which creates an appropriate external microenvironment for recovery of the spinal cord. However, the approaches of surgery cannot provide further driving forces for SCI recovery [12,20]. In addition, non-surgical procedures, such as drugs, hyperbaric oxygen, and hypothermia, have shown specific efficacy in the treatment of SCI, but they have apparent shortcomings [21]. For ex- ample, the U.S. Food and Drug Administration has approved methyl- prednisolone (MP) for the treatment of SCI. MP can inhibit secondary injury by stabilizing cell membranes, inhibiting the release of endor- phins, and limiting inflammation. However, when used in high doses, it can also cause side effects, including abnormal autonomic reflexes, immunosuppression, deep vein thrombosis, and pulmonary embolism [22,23]. Furthermore, there has recently been a growing suspicion about the role of MP in promoting the recovery of nerve function during SCI therapy [24,25]. Both hypothermia and hyperbaric oxygen are aux- iliary therapy measures with limited efficacies and complicated pro- cesses [26,27]. Therefore, an effective strategy is urgently required for SCI therapy. 3 Y. Luo, F. Xue, K. Liu et al. Thus far, studies on SCI treatment have mainly focused on stem cell therapy, drug screening, gene therapy, tissue engineering, and their bi- ological mechanisms [28,29]. Particularly, tissue engineering has irre- placeable advantages in SCI therapy. The wound can be bridged, the formation of nerve scars can be inhibited, and axons at both ends of the injury can be physically guided to reconnect by tissue engineering scaffolds. Moreover, to improve the repair effect of injured spinal cord, tissue engineering scaffolds can be used as carriers of bioactive compo- nents or cells to form biofunctional scaffolds to inhibit secondary dam- age or provide the driving force for nerve regeneration [30,31]. As a result of advances in materials science and bioscience, re- searchers have developed various unique insights into the application of tissue engineering scaffolds for SCI repair and have predicted their clinical applications [32]. Several studies have confirmed the advan- tages of neural tissue engineering scaffolds for SCI therapy [33,34]. The internal structures of scaffolds not only provide adequate space for cell survival and material exchange in the nervous system, but they also offer physical guidance for axon growth to some extent. The mechanical performance of the scaffold can compensate for the lack of mechanical support caused by SCI. The conductivity of the scaffold can simulate the electrophysiological microenvironments of the nerve tissue and transmit endogenous bioelectrical signals [35]. Furthermore, the bio- logical function of the scaffold can specifically overcome adverse pathophysiological changes to regulate the microenvironments and en- dogenous neural stem cells (NSCs), promoting SCI repair. 2. Physical support of scaffolds for spinal cord injury repair Tissue engineering scaffolds provide a favorable growth microenvi- ronment and an instructional cue for nerve regeneration during SCI re- pair, which, to some extent, depends on the physical support of scaffolds [36]. Physical support suitable for SCI is a prerequisite of nerve repair, mainly depending on utilized materials and techniques during the prep- aration. Currently, various natural and synthetic polymers have been used in scaffold manufacturing. Natural polymers, including hyaluronic acid (HA) [37,38], chitosan (CS) [39-42], collagen (Col) [43,44], agarose [45], alginate [46], gelatin (Gel) [47,48], and acellular matrix [49], have been used to research nerve regeneration in SCI. Synthetic polymers in- clude poly(lactic-co-glycolic acid) (PLGA) [50,51], poly(ε-caprolactone) (PCL) [52], poly(lactic acid) (PLA) [47,53], poly(ethylene glycol) (PEG) [42,54], hydroxypropyl methacrylamide (HPMA) [55], poly(sialic acid) (PSA), methacrylate (MA) [48], poly(3,4-ethylene dioxythiophene) (PEDOT) [56], polypyrrole (PPy) [57–59], and polyaniline (PANI) [60]. The properties and applications of polymers have been discussed in de- tail in our previous review [61]. Current applications of polymers often involve combinations of mul- tiple polymers as it is challenging to provide the required physical sup- port for damaged spinal cords when using a single polymer. To prepare customized scaffolds with various forms, structures, and functions, many technologies, including electrospinning and three-dimensional (3D) printing, have been applied for the preparation [62,63]. In addition to biocompatibility and biodegradability, the internal structures, me- chanical properties, and electrical conductivities of scaffolds are also sig- nificant physical properties to ensure the best repair effect of SCI. 2.1. Biocompatibility and biodegradability Biocompatibility refers to the compatibility performance of living tissue in response to non-active materials. Implantation of foreign ma- terials into the body will affect the biological tissue microenvironments, and biological tissues will also affect the implanted materials [64]. The interaction between the implant and biological tissue will keep for a long time until a state of equilibrium is reached, or the implant is removed. Biocompatibility is necessary for the application of tissue en- gineering scaffolds in the medical field and a prerequisite for satisfac- tory repair effects because the physiological activities of cells, such as 4 Materials and Design 201 (2021) 109484 survival, proliferation, migration, and differentiation, are based on this characteristic [65,66]. Notably, during secondary injury following SCI, inflammatory cells accumulate at the site of injury and secrete large quantities of inflammatory factors, such as interleukin-1ẞ (IL-1ẞ), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-α), thus forming an inflammatory microenvironment [1,18]. In this case, scaffolds implanted into the damaged spinal cord need to have better biocompatibility. In order to meet this condition, both natural and synthetic polymers currently used to prepare scaffolds for SCI repair need to be tested for toxicity at the cell and tissue levels. In addition to polymers, the liquid used in the preparation process is also an essential factor affecting the biocompatibility of scaffolds. Toxic organic liquids, such as methylene chloride, trichloromethane, and tetrahydrofuran, are used as solvents or crosslinking agents to prepare scaffolds through electrospinning or 3D printing [67]. Although organic solvents can be removed by natural volatilization, vacuum drying, dialysis, freeze-drying or other methods, it is still quite challenging to remove them thoroughly. Therefore, re- searchers should make a long-term effort to explore strategies that can effectively remove toxic liquids. The biodegradation of scaffolds is another crucial factor in tissue re- pair. The ideal biodegradation rate can provide adaptive growth space and mechanical support for newborn tissues. In general, the perfect biodegradation rate of scaffolds mainly depends on the growth rate of tissues [68]. Currently, there is no specific standard for the degradation rates of scaffolds for SCI repair, with degradation times ranging from weeks to years. In the future, it is of great significance to explore the ac- curate degradation times of scaffolds for the best SCI repair effect. The degradation time of a scaffold can be controlled by using composite ma- terials. To prepare scaffolds with longer degradation times, CS and HA can be mixed to create satisfactory scaffolds. Similarly, mixtures of PLGA and poly(L-lysine) [51], CS CS and Col [41], and Gel and PLA [69] have been used to prepare scaffolds with appropriate degradation rates. Li et al. designed an injectable and biodegradable hybrid hydrogel for application in SCI models. Degradation tests in vivo and in vitro showed that the degradation times of scaffolds were at least two weeks (Fig. 2A, B, and D), and no abnormal inflammatory response was found in tissues around scaffolds (Fig. 2C). Besides, the hydrogel had no significant cytotoxicity in vitro (Fig. 2E and F) [53]. 2.2. Internal structure The internal structure of the scaffold can provide channels for the ex- change of metabolic substances. Additionally, it can guide the regenera- tion of nerve tissue through physical contact [70]. Researchers have attempted to create a scaffold with an internal structure similar to that of the spinal cord to provide a natural path for tissue growth [71,72]. Previously, the acellular matrix of neural tissue was considered as one of the most promising scaffolds because it can retain the major internal structure of nerve tissue. However, its application was signifi- cantly limited, owing to limited supply and immune rejection [73,74]. Many studies consider porosity, aperture, and specific morphology, such as orientation arrangement, to be the most essential factors of the internal structures for SCI repair. Particularly, scaffolds with a poros- ity of 90% and aperture of 10-100 μm are the most suitable scaffolds for cell growth, and the directional arrangement structure can guide the or- derly growth of nerve fiber to the maximum extent [75,76]. Currently, technologies, including freeze-drying, electrospinning [77], and 3D printing [62], are used to fabricate scaffolds with suitable internal struc- tures. Although freeze-drying can control the porosity and aperture of scaffolds, the prepared scaffolds usually have the disadvantages of in- sufficient mechanical integrity and non-orientation. Electrospinning is widely used to prepare oriented scaffolds by forming oriented fibers. However, electrospun scaffolds are mainly used to treat peripheral nerve injury and are rarely used for SCI repair. This may be due to the Y. Luo, F. Xue, K. Liu et al. A Day 1 Day 3 Day 7 Day 14 C Day 1 Materials and Design 201 (2021) 109484 Day 3 0.2 0.4 0.6 0.8 B Day 7 Day 14 Day 1 D 40 Weight loss (%) Day 3 Day 35 55 30 25 25 4°C 37 °C 20 20 15 ヨ Cell viability (%) Day 14 100 80 60 60 40 40 20 NIH-3T3 F Astrocyte Cell viability (%) 50 50 40 40 30 50 20 20 10 10 50 μm 10 2 3 4 Degradation time (week) 0 25 50 100 200 400 600 800 1000 Copolymer concentration (μg mL-1) 0 0 1 2 4 6 8 10 Concentration of CAB (nM) Fig. 2. Biodegradation and biocompatibility of cabazitaxel (CAB)-encapsulated hybrid hydrogel. (A) Representative fluorescence imaging of mice after subcutaneous injection of hydrogel. (B) A representative optical image of in vivo degradation time of mixed hydrogel. (C) Hematoxylin and eosin (H&E) staining was performed on the tissue around the injected hydrogel at a predetermined time point, and results demonstrated that the hydrogel had good biocompatibility. (D) Degradation rate of hydrogels at different temperatures. The degradation rate was faster at 37 °C compared to that at 4 °C. (E) and (F) The cytotoxicity of the polymer and CAB were tested. Reproduced with permission from [53]. A Lesion site Grey matter White matter B Cooing unit Heating unit C Rat spinal cord Top view Side view Growth Scaffold Cells A+B Cells B Cells A Fig. 3. Preparation of 3D printed scaffold with similar structure of spinal cord. (A) Schematic illustration of spinal cord illustrating grey matter and white matter boundaries and a design for a 3D bioprinted multichannel scaffold for modelingspinal cord. (B) Schematic diagram of 3D printing. (C) Exhibition of a transected rat spinal cord and design principle for scaffolds consisting of multiple and continuous channels. Top view and side view image of scaffold. Reproduced with permission from [81]. 5See Answer
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