Polymers in Therapeutic Delivery

Description

The delivery of genes and drugs remains an active research area, with new challenges arising from the need to deliver specialized cargo, including antibodies, peptides, proteins and oligonucleotides and the need of reaching desired release profiles. Polymer-based systems hold great promise, as polymers can be designed and modified to meet new challenges. Frontline investigators describe polymer systems, such as polyethylenimine, poly(thioether anhydrides), stimuli-responsive hydrogels, and metal-organic nanomaterials, to deliver specific therapeutics, such as genetic materials and anticancer drugs, in controlled manners. Polymer chemists, pharmaceutical scientists, material chemists, and other researchers will find this collection valuable for their research. Polymers in Therapeutic Delivery ACS Symposium Series1350 Polymers in Therapeutic Delivery Library of Congress Cataloging-in-Publication Data Foreword Preface A Polymer Physics Perspective on Why PEI Is an Effective Nonviral Gene Delivery Vector Comparison of In Vitro Performances of Nanorod and Nanofiber Polyplexes Prepared from Plasmid DNA and Poly(L-lysine) Terminally Bearing Multi-Arm PEG A Novel Polysaccharide Carrier for Targeted Delivery of Therapeutic Oligonucleotides to β-Glucan Receptors Sustained Drug-Releasing Systems Using Temperature-Responsive Injectable Polymers Containing Liposomes Design of Stimuli-Responsive Polyampholytes and Their Transformation into Micro-Hydrogels for Drug Delivery Cellular Delivery of Hoechst 33342 Anticancer Drug from Crosslinked Poly(thioether anhydrides): A Cytotoxicity and Efficacy Study Metal–Organic Nanomaterials for Drug Delivery Editors’ Biographies Indexes Indexes Author Index Subject Index Preface 1 A Polymer Physics Perspective on Why PEI Is an Effective Nonviral Gene Delivery Vector Introduction Protonation of Diamines Figure 1. The difference in two pKas for the diamines separated by different numbers of carbon atoms. The symbols are experimental data reported in Bencini et al. The horizontal dashed line shows the limit of ΔpK due to statistic effect when the additional cost of free energy of ionization becomes zero. The solid line is the calculated ΔpK according to eqs 1 and 2 by using the distance r measured in molecular models 38. Protonation of PEI Chains Figure 2. Overlay of experimental titration curves from Smits et al. 39 (shown in symbols) with computational titration curves (shown as lines) in the distance-dependent dielectric model. Salt concentrations for experimental curves are Cs = 0 M (circles) and 0.1 M (triangles). Salt concentrations for computational titration curves are Cs = 0 M (dot-dashed line), 0.01 M (solid line), and 0.1 M (dashed line). The pK0 was assumed to be 10.0 in computational titration curves. Reproduced with permission from reference 38. Copyright 2010 American Chemical Society. Conformational Change of PEI Chain at a Different pH Figure 3. Visualizations of PEI chains for the fraction of protonated amines α: (a) α = 0, (b) α = 0.25, (c) α = 0.5, (d) α = 0.75, and (e) α = 1.0. In order to see monomeric structure of the chain, the images are not to scale. Reproduced with permission from reference 41. Copyright 2019 American Chemical Society. Figure 4. Panel (a) is the average distance between neighboring amines, L0; panel (b) is the population of the torsional angle (defined by N-C-C-N) in a gauche state. The legend in panel (b) is the salt concentrations. All data are obtained from molecular dynamic simulations of a single PEI chain. Adapted with permission from reference 41. Copyright 2019 American Chemical Society. Figure 5. Change in (a) radius of gyration Rg (triangles) and end-to-end distance r (squares) and (b) persistence length Lp for a single PEI chain with 40 repeating units as a function of protonation level, α, for simulations in salt-free conditions. Reproduced with permission from reference 41. Copyright 2019 American Chemical Society. Ion Condensation Around the PEI Chain at a Highly Charged Regime Figure 6. (a) N–Cl- radial distributions for α = 0, 0.25, 0.5, and 1.0 as shown in the figure legend, with a ~ 1 M NaCl concentration. The α values of 0, 0.25, 0.5, and 1 are represented by the red, blue, green, and purple lines respectively. (b) Excess g(r) is defined as the integral of the radial distribution above the bulk concentration: . The integration ranges from 2.85 Å, the distance of closest approach between N and Cl-, to 20 Å, where g(r) approaches 1, the bulk ion density, for all protonation states. Reproduced with permission from reference 41. Copyright 2019 American Chemical Society. Emerged Physical Picture of Potential PEI Actions During Delivery References 2 Comparison of In Vitro Performances of Nanorod and Nanofiber Polyplexes Prepared from Plasmid DNA and Poly(L-lysine) Terminally Bearing Multi-Arm PEG Introduction Scheme 1. Schematic image of multi-arm PEGylated poly(L-lysine) (maPEG-PLL). Experimental Section Materials Preparation of Nanorod and Nanofiber Polyplexes Cellular Uptake and Transfection of Polyplexes Interaction of Polyplexes with Heparin Reactivity of Polymerase to pDNA in Polyplexes Results and Discussion Figure 1. Cellular uptake nanorod (black) and nanofiber (gray) polyplexes prepared using FITC-labeled pDNA by HeLa cells was evaluated by flow cytometry (A). Luciferase activity of HeLa cells treated with nanorod and nanofiber polyplexes with and without chloroquine (B). In (A), white histogram is nontreated cells. In (B), mean ± s.d. (n = 3). Figure 2. Electrophoretic evaluation of nanofiber polyplexes (A) and nanorod polyplexes (B) tolerability against an exchange reaction with heparin. Figure 3. Change in Ct values of naked pDNA, nanorod polyplexes, and nanofiber polyplexes with pDNA copy number (A) and PCR efficiency of nanorod and nanofiber polyplexes (B). In (A) and (B), mean ± s.d. (n = 3). Figure 4. PCR efficiency of naked pDNA (A), nanofiber polyplexes (B), and nanorod polyplexes(C) in the presence of crowding cosolutes including Ficoll and dextran. Conclusion Acknowledgments References 3 A Novel Polysaccharide Carrier for Targeted Delivery of Therapeutic Oligonucleotides to β-Glucan Receptors Introduction Figure 1. The chemical structure of SPG (A) and ODN/SPG complex (B). Use of ODN/SPG Complexes in Targeted Delivery of Therapeutic ODNs Recognition of ODN/SPG Complexes by the β-1,3-D-Glucan Receptor Dectin-1 Figure 2. Time courses of frequency changes of a QCM coated with immobilized rDectin-1 in response to the addition of (A) t-SPG and r-SPG, (B) PS-dA60, and PS-dA60/SPG at 30 mg/mL. Reproduced with permission from reference 18. Copyright 2014 Elsevier. Treatment with an SPG/ODN Complex Can Protect Mice from Induced Hepatitis Figure 3. (A) Images of electrophoresis gels after mixing of FITC-AS-TNF-α and Cy5-labeled sense TNF-α RNAs. The sense TNF-α RNA was incubated with FITC-AS-TNF-α and FITC-AS-TNF-α/SPG in PBS for 2 h at 37 °C. Cy5 from RNA and FITC from AS-TNF-α were observed. The arrow indicates the band for sense TNF-α RNA hybridized with AS-TNF-α/SPG. (B) Silencing of TNF-α secretion determined by enzyme-linked immunosorbent (immunoadsorbent) assay following administration of AS-TNF-α to LPS-stimulated peritoneal macrophages for various dose conditions, showing an AS-TNF-α dose dependence of the silencing. (C) AS-TNF-α/SPG-mediated TNF-α inhibition of the progression of LPS/D-GalN-induced fulminant hepatitis. The fluorescence intensities of cell lysates of liver PCs and NPCs were measured 30 min after incubation with 0.5 mm FITC-labeled AS-TNF-α/SPG or AS-TNF-α/SPG. (D) The TNF-α level in the serum was measured at 1 h after administration of LPS/D-GalN. The blood was obtained by retroorbital bleeding; n and c represent administrations of naked AS-TNF-α and AS-TNF-α/SPG, respectively. *P < 0.05, **P < 0.01. (E) Representative liver histologies of LPS/D-GalN-induced hepatitis in mice injected with PBS, AS-TNF-α, or AS-TNF-α/SPG (5 mg/kg). At 6 h after LPS/d-GalN injection, the livers were stained with hematoxylin and eosin (H & E). Original magnification × 200. Reproduced with permission from reference 9. Copyright 2011 Elsevier. An Anticancer SPG Complex Is Internalized to Cytosol and Suppresses Cell Growth Figure 4. (A) Cell viability after treatment with AS-YB-1(014)/SPG at 0.4 or 1.0 mm for PC-9 cells. Results are presented as means ±SD (n = 3). *P < 0.01. (B) Schematic illustration of the protocol for the preparation of the template containing AS-YB-1(014). PC-9 cells were treated with AS-YB-1(014)/SPG at 1 μm and incubated for the indicated time. The endosome and cytoplasm components were then collected by centrifugation and mixed with biotin-adaptor-DNA. After addition of Dynabeads Streptavidin, the AS-YB-1(014) bound to biotin-DNA was purified, and the adaptor-DNA bound to AS-YB-1(014) was dissociated by heating at 96 °C for 5 min and by using Dynabeads system (Thermo Scientific). After the ligation of AS-YB-1(014) to linker-DNA by using T4 DNA ligase, the copy numbers of AS-YB-1(014) were determined by RT-PCR. (C) Quantification of the AS-YB-1(014) copy number in the endosomal (upper) and cytoplasmic (lower) components at the indicated times after treatment with the AS-YB-1(014)/SPG complex. Results are presented as means ±SD (n = 2) (D) Schematic illustration of the RT-PCR protocol using AS-YB-1(014) as a primer for reverse transcription. PC-9 cells were treated with AS-YB-1(014)/SPG at 1 μm and incubated for 24 h. After the cytoplasm component was collected by centrifugation, it was mixed with biotin-DNA. Dynabeads Streptavidin was then added, and the mRNA (YB-1) bound to biotin-DNA was purified and subjected to RT-PCR. (E) The level of amplification of YB-1 using the dA40/SPG or AS014/SPG complex after treatment with or without Dynabeads Streptavidin. M: DNA Ladder Mix. Reproduced with permission from reference 34. Copyright 2019 Springer Nature. Summary References 4 Sustained Drug-Releasing Systems Using Temperature-Responsive Injectable Polymers Containing Liposomes Introduction Figure 1. Design of sustained drug-releasing system using biodegradable IP and drug-loaded liposome. Experimental Section Materials Preparation of Liposomes Preparation of IP Formulation Rheological Measurements In Vitro Release Test In Vivo Experiments Results and Discussion Figure 2. Fluorescence spectra for pyranine and DPX (quencher)-loaded liposome dispersion (open circles), liposome–IP after gelation and cooling (open diamonds), and liposome–IP after the addition of Triton X-100 (final concentration: 4.8 vol %) (closed diamonds). Figure 3. Storage moduli (G′) and loss moduli (G′′) of (A) 25wt% tri-PCG solution 225 μL + 75 μL PBS, (B) 25wt% tri-PCG solution 225 μL + 75 μL liposome (100 nm) solution (lipid concentration: 50 mM) as a function of temperature. Figure 4. (A) In vitro pyranine release from IP hydrogel containing free pyranine (pyranine-IP, open diamonds), pyranine-liposome-IP containing 50 nm (closed triangle) or 100 nm (closed circle) liposomes. (B) Lipid release from pyranine-liposome-IP containing 50 nm (closed triangle) or 100 nm (closed circle) liposomes. Liposomes were labeled with 0.1mol% Rh-PE. Figure 5. Fluorescence images of mice injected with pyranine, pyranine-loaded liposome, IP formulation containing free pyranine, or IP formulation containing pyranine-loaded liposome. The samples were subcutaneously injected to BALB/c mice backs (n = 5). Pyranine fluorescence was detected from mice backs using IVIS system. Red color represents strong signal of pyranine. Excitation and detection wavelengths were 460 and 520 nm, respectively. Figure 6. Quantification of the fluorescence intensity for mice at the injected sites (n = 4). In pyranine–IP-treated groups, the fluorescence signal was the same as the background signal after two days. Figure 7. Plasma concentration of pyranine in mice (n=4) for 30 days after subcutaneous injection of free pyranine solution (closed triangle), pyranine-IP ( open diamonds), pyranine-liposome (closed circles), and pyranine-liposome-IP (closed diamonds). Dotted line represents the limit of detection (0.0116 mg/L). *p < 0.01 vs. other groups. Conclusion Acknowledgments Appendix Figure A1. Release of pyranine from pyranine- and DPX-loaded liposomes with different diameters incubated in PBS at 37 °C. References 5 Design of Stimuli-Responsive Polyampholytes and Their Transformation into Micro-Hydrogels for Drug Delivery Introduction Materials and Methods Synthesis of Polyampholytes Figure 1. Synthesis pathway of PLL-SA50, PLL-BSA50, PLL-DMGA50, and PLL-PA50. Characterization of Polyampholytes Phase Separation of Polyampholytes (Turbidity Measurements) Preparation of Hydrogel Particle Size and Morphology of Polyampholytes Encapsulation and Release Profiles of Polyampholyte Hydrogels Encapsulation Model Drug (Curcumin) Figure 2. Schematic illustration of preparation of curcumin-encapsulated polyampholyte hydrogels (PAL@Cur hydrogels). Release Profile of Polyampholyte Hydrogels Cytotoxicity Assay Results and Discussion Synthesis of Polyampholytes Figure 3. 1H NMR spectra of (a) PLL, (b) PLL-SA50, (c) PLL-BSA50, (d) PLL-DMGA50, and (e) PLL-PA50 in D2O. Phase Separation of Polyampholytes Effect of Polymer Concentration Figure 4. Phase separation behavior of polyampholytes in aqueous solutions. Temperature dependence of transmittance at various polyampholyte concentrations: (a) PLL-SA50, (b) PLL-BSA50, (c) PLL-DMGA50, and (d) PLL-PA50. (e) Phase diagram of the polyampholyte solutions. Effect of Concentration of Salt (Ionic Strength) Figure 5. Phase separation behavior of polyampholytes in aqueous solutions (10%). Temperature dependence of transmittance with various concentration of salt: (a) PLL-SA50, (b) PLL-BSA50, (c) PLL-DMGA50, and (d) PLL-PA50. Salt concentrations used are indicated next to the curves. Effect of pH Figure 6. Phase separation behavior of polyampholytes in aqueous solutions (10%). (a) pH dependence of transmittance at various pH values. Schematic illustration of the proposed mechanism of pH-dependent phase separation of (b) PLL-SA50 or PLL-DMGA50, and (c) PLL-BSA50 or PLL-PA50. Figure 7. Phase separation behavior of polyampholytes in aqueous solutions. pH dependence of transmittance with various pH of (a) PLL-PA45 and (b) PLL-PA35. Particles Size and Morphology of Polyampholytes Figure 8. SEM images of polyampholyte hydrogels: (a) PLL-SA50, (b) PLL-DMGA50, (c) PLL-PA50, (d) PLL-BSA50, and (e) enlarged view of PLL-BSA50. Encapsulation and Release Profiles of Polyampholyte Hydrogels Figure 9. Calibration curve of curcumin; (a) UV–vis spectra of curcumin at various concentrations and (b) plot of absorbance with various concentrations of curcumin. Figure 10. Release profiles of curcumin from (a) PLL-SA50 and (b) PLL-PA50 hydrogels at different pH. Figure 11. Schematic illustration for the pH-triggered drug release with polyampholyte hydrogels. Cytotoxicity Assay Figure 12. Cytotoxicity of PLL-SA50, PLL-BSA50, PLL-DMGA50, and PLL-PA50 with normal cell L929. Conclusions References 6 Cellular Delivery of Hoechst 33342 Anticancer Drug from Crosslinked Poly(thioether anhydrides): A Cytotoxicity and Efficacy Study Introduction Scheme 1. Idealized schematic of surface erosion and bulk erosion. Changes in shading indicate changes in polymer properties. Scheme 2. Synthesis of polyanhydrides based on sebacic acid and 1,3-bis(p-carboxyphenoxy)propane. Scheme 3. Radical-mediated thiol–ene polymerization used to synthesize crosslinked poly(thioether anhydrides) (PAHs), and their subsequent hydrolysis and degradation. Adapted with permission from refs. 46 and 47. Copyright 2014 & 2015 American Chemical Society. Figure 1. Erosion, expressed as % mass remaining for PAHs made by radical-mediated thiol–ene polymerizations of 4-pentenoic anhydride (PNA) and pentaerythritol tetrakis (3-mercaptopropionate) (PETMP), containing no dye or 1 wt % dye (○ and ×, respectively) and the dye release profile (□, 1 wt % dye). Adapted with permission from ref. 47. Copyright 2014 American Chemical Society. Figure 2. Summary of spectral (infrared, IR), thermal, and physical properties (from dynamic mechanical analysis, DMA) of PAHs made from the radical polymerization of PNA and PETMP. Adapted with permission from ref. 44. Copyright 2010 American Chemical Society. Scheme 4. Structure of H33342. Experimental Section Materials Polymer Synthesis Intercellular Delivery and Efficacy of H33342 Results and Discussion Figure 3. Intracellular delivery and uptake of H33342, evaluated by fluorescence microscopy after 36 h of exposure. Representative images of H33342 fluorescence in A-375 melanoma cells are shown: (A) control conditions, (B) +1000 mg/L PAH, (C) +75 μM H33342, (D) +75 μM H33342–1000 mg/L PAH. Arrows point to apoptotic cells showing nuclear condensation or fragmentation. Figure 4. Effect of H33342’s encapsulation into PAH on cytotoxic activity and subsequent A-375 cell death using fluorescence microscopy and PI exclusion assay. Images collected after 24 h of exposure to A-375 cells showing (A) control conditions, (B) 1000 mg/L PAH, (C) 10% ethanol (EtoH) compared with cells treated with (D) 75 μM H33342 and (E) 75 μM H33342–1000 mg/L PAH. Blue and red nuclear stains indicate live cells containing H33342 and dead cells, respectively. Figure 5. Time course study for intracellular delivery of 75 μM H33342 to A-375 cells for untreated A-375 cells, +1000 mg/L PAH, +free 75 μM H33342, and +75 μM H33342–1000 mg/L PAH. Images recorded at 1, 6, 24, 36, and 48 h after incubation. Blue and red nuclear stains indicate live and dead cells, respectively. Figure 6. Time course study for intracellular delivery of 16 μM H33342 to A-375 cells (untreated A-375 cells, +1000 mg/L PAH, free 16 μM H33342, +16 μM H33342–1000 mg/L PAH). Images are recorded at 1, 6, 24, 36, and 48 h after incubation. Blue and red nuclear stains indicate live and dead cells, respectively. Figure 7. Cytotoxic effects of H33342 on cell viability of A-375 cells after exposure to (A) 75 μM H33342 and (B) 16 μM H33342, with respect to various controls. Conclusions Acknowledgments References 7 Metal–Organic Nanomaterials for Drug Delivery Introduction Methods for Preparing MONs Solvo/Hydrothermal Synthesis Ultrasound-Assisted Synthesis Microwave-Assisted Synthesis Mechanochemical Synthesis Reverse-Phase Emulsion Method SMILE Method Strategies for Controlling the Particle Size of MONs Figure 1. LaMer model of the nanoparticle prenucleation (I), nucleation (II), and particle growth process (III). Controlling Nucleation and Particle Growth Generating Nanoreactors Figure 2. Synthesis of MONs with nanoreactors. (A) Synthesis of ZIF-8 MONs with emulsions as the nanoreactor. Reproduced with permission from reference 18. Copyright 2014 American Chemical Society. (B) Synthesis of Cu(DDC)2 NPs with liposomes as the nanoreactor 22. Continuous Production Figure 3. Continuous production of MONs. (A) General flow reactor setup for the production of MONs. Reproduced with permission from reference 34. Copyright 2014 Springer Nature. (B) Continuous production of Cu(DDC)2 MONs with a 3D-printed microfluidic device. Reproduced with permission from reference 36. Copyright 2019 Elsevier. Surface Functionalization of MONs Figure 4. Surface modification of MONs. (A) Modification of alkyne-functionalized CuMOP through azide substituted organic bridging linkers [4-azidobenzoate (4-N3B−) and 5-azidoisophthalate (5-N3IP−2)] via covalent bonds (click reaction). Reproduced with permission from reference 37. Copyright 2017 Ahmad et al. under CC-BY 4.0 license. (B) Modification of MONs with noncovalent interactions (metal–His-tags coordination interactions). Reproduced with permission from reference 40. Copyright 2017 American Chemical Society. Covalent Modifications Noncovalent Modifications Stimulus-Responsive MONs as Smart Drug Delivery Systems Figure 5. Different stimulus-responsive MONs. (A) PAA@ZIF-8 NPs as pH-responsive MONs. Reproduced with permission from reference 43. Copyright 2014 The Royal Society of Chemistry. (B) Fe3O4@PDA as magnetic-responsive MONs. Reproduced with permission from reference 51. Copyright 2019 Chen et al. under CC-BY 4.0 license. (C) MOF-74-Fe(II) as ion-responsive MONs. Reproduced with permission from reference 57. Copyright 2014 American Chemical Society. (D) PNIPAM-modified MOF as thermal-responsive MONs. Reproduced with permission from reference 58. Copyright 2015 The Royal Society of Chemistry. pH-Sensitive MONs Magnetic-Responsive MONs Ion-Responsive MONs Thermal-Sensitive MONs Bioactive MONs as Therapeutics Conclusions and Future Perspectives Acknowledgments References Editors’ Biographies Tomoko Fujiwara X. Michael Liu Yuichi Ohya Yongmei Wang Indexes Author Index Subject Index D H I M N P T