Recent progress in polymeric gene vectors: Delivery mechanisms, molecular designs, and applications.

Gene therapy and gene delivery have drawn extensive attention in recent years especially when the COVID-19 mRNA vaccines were developed to prevent severe symptoms caused by the corona virus. Delivering genes, such as DNA and RNA into cells, is the crucial step for successful gene therapy and remains a bottleneck. To address this issue, vehicles (vectors) that can load and deliver genes into cells are developed, including viral and non-viral vectors. Although viral gene vectors have considerable transfection efficiency and lipid-based gene vectors become popular since the application of COVID-19 vaccines, their potential issues including immunologic and biological safety concerns limited their applications. Alternatively, polymeric gene vectors are safer, cheaper, and more versatile compared to viral and lipid-based vectors. In recent years, various polymeric gene vectors with well-designed molecules were developed, achieving either high transfection efficiency or showing advantages in certain applications. In this review, we summarize the recent progress in polymeric gene vectors including the transfection mechanisms, molecular designs, and biomedical applications. Commercially available polymeric gene vectors/reagents are also introduced. Researchers in this field have never stopped seeking safe and efficient polymeric gene vectors via rational molecular designs and biomedical evaluations. The achievements in recent years have significantly accelerated the progress of polymeric gene vectors toward clinical applications.

Delivery of CRISPR/Cas9 Plasmid DNA by Hyperbranched Polymeric Nanoparticles Enables Efficient Gene Editing.

Gene editing nucleases such as CRISPR/Cas9 have enabled efficient and precise gene editing in vitro and hold promise of eventually achieving in vivo gene editing based therapy. However, a major challenge for their use is the lack of a safe and effective virus-free system to deliver gene editing nuclease elements. Polymers are a promising class of delivery vehicle due to their higher safety compared to currently used viral vectors, but polymers suffer from lower transfection efficiency. Polymeric vectors have been used for small nucleotide delivery but have yet to be used successfully with plasmid DNA (pDNA), which is often several hundred times larger than small nucleotides, presenting an engineering challenge. To address this, we extended our previously reported hyperbranched polymer (HP) delivery system for pDNA delivery by synthesizing several variants of HPs: HP-800, HP-1.8K, HP-10K, HP-25K. We demonstrate that all HPs have low toxicity in various cultured cells, with HP-25K being the most efficient at packaging and delivering pDNA. Importantly, HP-25K mediated delivery of CRISPR/Cas9 pDNA resulted in higher gene-editing rates than all other HPs and Lipofectamine at several clinically significant loci in different cell types. Consistently, HP-25K also led to more robust base editing when delivering the CRISPR base editor "BE4-max" pDNA to cells compared with Lipofectamine. The present work demonstrates that HP nanoparticles represent a promising class of vehicle for the non-viral delivery of pDNA towards the clinical application of gene-editing therapy.

Scaffolds with controlled release of pro-mineralization exosomes to promote craniofacial bone healing without cell transplantation.

Biomimetic bone regeneration methods which demonstrate both clinical and manufacturing feasibility, as alternatives to autogenic or allogenic bone grafting, remain a challenge to the field of tissue engineering. Here, we report the pro-osteogenic capacity of exosomes derived from human dental pulp stem cells (hDPSCs) to facilitate bone marrow stromal cell (BMSC) differentiation and mineralization. To support their delivery, we engineered a biodegradable polymer delivery platform to improve the encapsulation and the controlled release of exosomes on a tunable time scale from poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) triblock copolymer microspheres. Our delivery platform integrates within three-dimensional tissue engineering scaffolds to enable a straightforward surgical insertion into a mouse calvarial defect. We demonstrate the osteogenic potential of these functional constructs in vitro and in vivo. Controlled release of osteogenic hDPSC-derived exosomes facilitates osteogenic differentiation of BMSCs, leading to mineralization to a degree which is comparable to exogenous administration of the same exosomes in human and mouse BMSCs. By recruiting endogenous cells to the defects and facilitating their differentiation, the controlled release of osteogenic exosomes from a tissue engineering scaffold demonstrates accelerated bone healing in vivo at 8 weeks. Exosomes recapitulate the advantageous properties of mesenchymal stem/progenitor cells, without manufacturing or immunogenic concerns associated with transplantation of exogenous cells. This biomaterial platform enables exosome-mediated bone regeneration in an efficacious and clinically relevant way.

Biodegradable nanofibrous temperature-responsive gelling microspheres for heart regeneration.

Myocardial infarction (heart attack) is the number one killer of heart patients. Existing treatments for heart attack do not address the underlying problem of cardiomyocyte (CM) loss and cannot regenerate the myocardium. Introducing exogenous cardiac cells is required for heart regeneration due to the lack of resident progenitor cells and very limited proliferative potential of adult CMs. Poor retention of transplanted cells is the critical bottleneck of heart regeneration. Here, we report the invention of a poly(l-lactic acid)-b-poly(ethylene glycol)-b-poly(N-Isopropylacrylamide) copolymer and its self-assembly into nanofibrous gelling microspheres (NF-GMS). The NF-GMS undergo thermally responsive transition to form not only a 3D hydrogel after injection in vivo, but also exhibit architectural and structural characteristics mimicking the native extracellular matrix (ECM) of nanofibrous proteins and gelling proteoglycans or polysaccharides. By integrating the ECM-mimicking features, injectable form, and the capability of maintaining 3D geometry after injection, the transplantation of hESC-derived CMs carried by NF-GMS led to a striking 10-fold graft size increase over direct CM injection in an infarcted rat model, which is the highest reported engraftment to date. Furthermore, NF-GMS carried CM transplantation dramatically reduced infarct size, enhanced integration of transplanted CMs, stimulated vascularization in the infarct zone, and led to a substantial recovery of cardiac function. The NF-GMS may also serve as advanced injectable and integrative biomaterials for cell/biomolecule delivery in a variety of biomedical applications.

Cationic polymeric N-halamines bind onto biofilms and inactivate adherent bacteria.

A series of amine-based cationic polymeric N-halamine precursors, poly(2,2,6,6-tetramethyl-4-piperidyl methacrylate-co-trimethyl-2-methacryloxyethylammonium chloride)(PMPQ), were synthesized by copolymerizing 2,2,6,6-tetramethyl-4-piperidyl methacrylate (TMPM) with trimethyl-2-methacryloxyethylammonium chloride (TMAC) at different molar ratios (TMPM:TMAC = 10:90,30:70,50:50,70:30, and 90:10). After chlorine bleach treatment, the TMPM moieties in the new copolymers were transformed into amine-based N-halamines (Cl-PMPQ). The chemical structures of the samples were characterized with 1H NMR, FT-IR, and UV spectra, and the molecular weights were determined by dynamic light scattering (DLS). With lower than 70 mol% of the original TMPM content, the resulting Cl-PMPQ copolymers were soluble in water, and demonstrated potent antibacterial functions against Escherichia coli (E. coli, a representative Gram-negative bacteria) and Staphylococcus epidermidis (S. epidermidis, a representative Gram-positive bacteria). E. coli and S. epidermidis were allowed to form biofilms on glass slides. Zeta potential analyses demonstrated that the Cl-PMPQ copolymers rapidly adsorbed onto the preexisting biofilms, and bacterial culturing studies confirmed that the bound Cl-PMPQ provided a total kill of the adherent bacteria in the biofilms. The kinetics of the Cl-PMPQ binding onto the preexisting biofilms were studied with UV analyses. The data fitted well to the bimodal model. The binding kinetic parameters of Cl-PMPQ onto the bacterial biofilms were thus determined.

Controlling bacterial fouling with polyurethane/N-halamine semi-interpenetrating polymer networks.

N -halamine-based interpenetrating polymer networks were developed as a simple and effective strategy in the preparation of antimicrobial polymers. An N-halamine monomer, N-chloro-2, 2, 6, 6-tetramethyl-4-piperidyl methacrylate, was incorporated into polyurethane in the presence of a cross-linker and an initiator. Post-polymerization of the monomers led to the formation of polyurethane/N-halamine semi-interpenetrating polymer networks. The presence of N-halamines in the semi-interpenetrating polymer networks was confirmed by attenuated total reflectance infrared, water contact angle, and energy-dispersive X-ray spectroscopy analysis. The N-halamine contents in the semi-interpenetrating polymer networks could be readily controlled by changing reaction conditions. The distribution of active chlorines within the semi-interpenetrating polymer networks was characterized with energy-dispersive X-ray spectroscopy. Contact mode antimicrobial tests, zone of inhibition studies, and scanning electron microscopy observations showed that the semi-interpenetrating polymer networks had potent antimicrobial and antifouling effects against both Gram-positive and Gram-negative bacteria. Release tests demonstrated the outstanding stability of the N-halamine structures in the new semi-interpenetrating polymer networks.

Doxorubicin loaded pH-responsive micelles capable of rapid intracellular drug release for potential tumor therapy.

Amphiphilic copolymers have been paid much attention for controlled drug release for many years due to their obvious advantages. In this study, an acid-triggered drug carrier system capable of rapid intracellular drug release is investigated for potential tumor therapy. The amphiphilic diblock copolymer poly(2-diisopropylaminoethyl methacrylate)-b-poly(2-aminoethyl methacrylate hydrochloride) (PDPA-b-PAMA) is prepared by atom transfer radical polymerization (ATRP). The molecular structure of the copolymer is confirmed by 1H NMR and gel permeation chromatography (GPC). The critical micelle concentration (CMC) value of the PDPA-b-PAMA is 0.005 mg/mL, which can ensure the thermodynamical stability of micelles even after significant dilution. The drug loading and encapsulation efficiencies of doxorubicin (DOX)-loaded micelles are 9.96% and 55.31%, respectively. Dynamic light scattering (DLS) and transmission electron microscope (TEM) show that the amphiphilic block copolymers self-assemble into spherical micelles with narrow polydispersity indexes (PDLs) at pH 7.4 and 6.8, but disassemble into random chain aggregations at pH 5.0. The DOX-loaded PDPA-b-PAMA shows obvious pH-responsive drug release profile when the pH value changes from 7.4 to 5.0, since it transforms from amphiphilicity to double hydrophilicity through the protonation of PDPA block (pK(a) - 6.2) in a relatively low pH condition, thus the loaded DOX can be rapidly released from the disassembling micelles. In addition, the micellar system also exhibits relatively low cytotoxicity and rapid drug release behaviour in tumor cells, which make it promising for tumor therapy.

Versatile functionalization of gene vectors via different types of zwitterionic betaine species for serum-tolerant transfection.

For ideal polymeric gene vectors, their serum stability is of crucial importance. Polycation vectors usually suffer from colloidal aggregation, which makes them easily cleared from the bloodstream. Recently, we reported a comb-shaped vector (DPD) consisting of a dextran backbone and disulfide-linked cationic poly((2-dimethyl amino)ethyl methacrylate) side chains for efficient gene delivery. In this work, versatile functionalization of DPD (as a model gene vector) was proposed via the introduction of different types of zwitterionic carboxybetaine and sulfobetaine species for improving biophysical properties. The incorporation of zwitterionic betaine did not destroy the DNA condensation capability of vectors. All the zwitterionic betaine-functionalized DPD vectors exhibited lower cytotoxicities than the pristine DPD. The DPD-b-polycarboxybetaine block copolymer (DPDbPC) exhibited better gene delivery abilities than the corresponding DPD-r-polycarboxybetaine random copolymer (DPDrPC). Moreover, in the serum case with a high concentration (30%) of fetal bovine serum, the DPD-b-polysulfobetaine block copolymer (DPDbPS) produced much higher gene transfection efficiencies than DPDbPC. Cellular internalization results indicated that the incorporation of zwitterionic betaine could benefit serum stabilities of vectors and enhance cellular uptake. The present study demonstrated that proper incorporation of zwitterionic betaine into gene carriers was an effective method to produce serum-tolerant transfection vectors.

Controlled drug release system based on cyclodextrin-conjugated poly(lactic acid)-b-poly(ethylene glycol) micelles.

Cyclodextrin-conjugated poly(lactic acid)-b-poly(ethylene glycol) (ß-CD-PLA-mPEG), a well-defined amphiphilic copolymer, was synthesized by controlled ring-open copolymerization and click coupling reaction, in order to obtain a biocompatible drug delivery system with controlled release profiles. The ß-CD-PLA-mPEG copolymer could self-assemble in aqueous solution to form micelles with a mean particle size of 173.4 nm, which will decrease to 159.2 nm after loaded with a kind of hydrophobic drug (indomethacin, IND). The IND-loaded ß-CD-PLA-mPEG micelles show spherical shape within the nano-size scale under TEM imaging. Compared with that formed by PLA-mPEG, the micelles formed by ß-CD-PLA-mPEG copolymer present higher drug loading efficiency and controlled release profile of IND, especially in the control of its initial burst release. Meanwhile, ß-CD-PLA-mPEG copolymer exhibits low toxicity to cells. The micelles formed by ß-CD-PLA-mPEG copolymer could be a promising controlled release system for various hydrophobic drugs.

Copolymer nanoparticles composed of sulfobetaine and poly(ε-caprolactone) as novel anticancer drug carriers.

Novel ABA type amphiphilic copolymers (PCL-APS-PCL) consisting of polycaprolactone (PCL) (A) as hydrophobic block and N,N'-bis (2-hydroxyethyl) methylamine ammonium propane sulfonate (APS) (B) as hydrophilic segment, self-assembled into nanoparticles (NPs) with solvent evaporation method. The sizes and size distributions of NPs were characterized by dynamic light scattering. The morphology of NPs was observed by scanning electron microscopy (SEM). The critical micelle concentration (CMC) was determined by fluorescent probe. The drug loading content (DLC) and the drug release amount were characterized by UV-visible spectrophotometer. The cytotoxicity of the NPs was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazoliumbromide (MTT) assay. It was found that the NPs were spherical in shape with sizes around 100 nm. The CMCs of the copolymers were quite low (×10(-4) mg/mL). The DLC decreased with lengthening of hydrophobic PCL block. In vitro drug release experiment demonstrated that the release rate of paclitaxel sped with the decrease of PCL length. MTT results showed that NPs were nontoxic to osteoblast and human epithelial carcinoma (hela) cells. After drug loading, NPs could restrain the growth of hela or even kill hela cells. Therefore, these preliminary studies suggest that the novel PCL-APS-PCL NPs have a great potential application as anticancer drug-delivery carriers.