Cationic Vectors Research Articles

Glutathione hybrid poly (beta-amino ester)-plasmid nanoparticles for enhancing gene delivery and biosafety

IntroductionCRISPR/Cas9 gene editing technology has significantly advanced gene therapy, with gene vectors being one of the key factors for its success. Poly (beta-amino ester) (PBAE), a distinguished non-viral cationic gene vector, is known to elevate intracellular reactive oxygen species (ROS) levels, which may cause cytotoxicity and, consequently, impact gene transfection efficacy (T.E.). ObjectivesTo develop a simple but efficient strategy to improve the gene delivery ability and biosafety of PBAE both in vivo and in vitro. MethodsWe used glutathione (GSH), a clinically utilized drug with capability to modulating intracellular ROS level, to prepare a hybrid system with PBAE-plasmid nanoparticles (NPs). This system was characterized by flow cytometry, RNA-seq, Polymerase Chain Reaction (PCR) and Sanger sequencing in vitro, and its safety and efficacy in vivo was evaluated by imaging, PCR, Sanger sequencing and histology analysis. ResultsThe particle size of GSH-PBAE-plasmid NPs were 168.31 nm with a ζ-potential of 15.21 mV. An enhancement in T.E. and gene editing efficiency, ranging from 10 % to 100 %, was observed compared to GSH-free PBAE-plasmid NPs in various cell lines. In vitro results proved that GSH-PBAE-plasmid NPs reduced intracellular ROS levels by 25 %–40 %, decreased the total number of upregulated/downregulated genes from 4,952 to 789, and significantly avoided the disturbance in gene expression related to cellular oxidative stress-response and cell growth regulation signaling pathway compared to PBAE-plasmid NPs. They also demonstrated lower impact on the cell cycle, slighter hemolysis, and higher cell viability after gene transfection. Furthermore, GSH hybrid PBAE-plasmid NPs exhibited superior safety and improved tumor suppression ability in an Epstein–Barr virus (EBV)-infected murine tumor model, via targeting cleavage the EBV related oncogene by delivering CRISPR/Cas9 gene editing system and down-regulating the expression levels. This simple but effective strategy is expected to promote clinical applications of non-viral vector gene delivery.

The impact of heparin and direct thrombin inhibitors on cell-penetrating polymer siRNA transfection.

Gene therapy using siRNA has become a promising strategy to achieve targeted gene knockdown for treatment of cardiovascular pathologies. However, efficient siRNA transfection often relies on cationic delivery vectors such as synthetic cell-penetrating polymers which are susceptible to interference by negatively charged molecules. Anticoagulants such as heparin, which is negatively charged and widely used in cardiovascular applications, may pose a significant barrier to effective siRNA delivery. We therefore conducted in vitro studies utilizing human smooth muscle and endothelial cells transfected with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β2-microglobulin (B2M) siRNA in the presence of heparin, argatroban, and bivalirudin in order to determine which anticoagulant therapy is most compatible for siRNA delivery. We observed that while heparin, at clinical doses, decreases the efficiency of siRNA targeted mRNA knockdown, mRNA knockdown is not inhibited in the presence of either argatroban or bivalirudin. Our data suggests that heparin should be avoided during siRNA therapy with cationic transfection agents, and argatroban and bivalirudin should be used in its stead.

A facile and scalable method to synthesize PEGylated PDMAEMA for gene delivery.

In recent years, cationic polymer vectors have been viewed as a promising method for delivering nucleic acids. With the advancement of synthetic polymer chemistry, we can control chemical structures and properties to enhance the efficacy of gene delivery. Herein, a facile, cost-effective, and scalable method was developed to synthesize PEGylated PDMAEMA polymers (PEO-PDMAEMA-PEO), where PEGylation could enable prolonged polyplexes circulation time in the blood stream. Two polymers of different molecular weights were synthesized, and polymer/eGFP polyplexes were prepared and characterized. The correlation between polymers' molecular weight and physicochemical properties (size and zeta potential) of polyplexes was investigated. Lipofectamine 2000, a commercial non-viral transfection reagent, was used as a standard control. PEO-PDMAEMA-PEO with higher molecular weight exhibited slightly better transfection efficiency than Lipofectamine 2000, and the cytotoxicity study proved that it could function as a safe gene vector. We believe that PEO-PDMAEMA-PEO could serve as a model to investigate more potential in the gene delivery area.

Abstract 3185: Glatiramer acetate enhances tumor retention and mitigates systemic toxicity of toll-like receptor 9 agonists as intratumoral immunotherapy

Abstract Pathogen-associated molecular patterns (PAMP) are conserved molecular structures produced by microorganisms that are recognized as foreign by receptors of the innate immune system. Unmethylated cytosine-guanine oligodeoxynucleotides (CpG ODNs) are a storied drug class from the PAMP family that ligates Toll-like receptor 9 (TLR9). CpG ODNs have shown promising antitumor effects in preclinical studies by inducing potent pro-inflammatory immune responses. However, there are still practical challenges that have hindered clinical success, including inefficient in vivo delivery and severe immune-related toxicities caused by systemic exposure to CpG. We previously identified that glatiramer acetate (GA), an FDA-approved, lysine-rich polypeptide, could be applied as a cationic drug delivery vector for CpG delivery proximal to the injection site while mitigating systemic release of pro-inflammatory cytokines. To dive into the mechanism and evaluate pharmacological efficacy in more pathological models that simulate colorectal carcinoma, we validated the biodistribution of GA-CpG complexes (or R4B) and explored their safety and antitumor immunity in multiple mouse colorectal carcinoma models. In the CT26 single tumor model, intratumoral R4B treatment displayed comparable antitumoral efficacy as CpG treatment. Genome-wide transcriptome analysis showed that R4B treatment elicited much more prominent immune system response pathways in both lesions and spleens. The biodistribution study proved that R4B localized and gradually released CpG around the injection site while ‘naked’ CpG diffused away from the injection site quickly. In the CT26 dual tumor-bearing model, intratumoral administration of R4B generated systemic immune efficacy that led to an abscopal effect on distant untreated cancers, comparable to treatment with CpG alone. Notably, R4B treatment accomplished these effects with greatly mitigated systemic pro-inflammatory cytokines compared to CpG alone. These data help further elucidate the noncanonical role of glatiramer acetate to serve as a nucleic acid delivery scaffold that potentiates the safety of CpG adjuvant for cancer immunotherapy. Citation Format: Huan Gong, Daniel J. Griffin, Chad E. Groer, Xiaoqing Wu, Moustafa M. Abdelaziz, Sa Wu, Liang Xu, Laird M. Forrest, Cory J. Berkland. Glatiramer acetate enhances tumor retention and mitigates systemic toxicity of toll-like receptor 9 agonists as intratumoral immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3185.

Intracellular Delivery of Functional Proteins with DNA-Protein Nanogels-Lipids Complex.

Using functional proteins for therapeutic purposes due to their high selectivity and/or catalytic properties can enable the control of various cellular processes; however, the transport of active proteins inside living cells remains a major challenge. In contrast, intracellular delivery of nucleic acids has become a routine method for a number of applications in gene therapy, genome editing, or immunization. Here we report a functionalizable platform constituting of DNA-protein nanogel carriers cross-linked through streptavidin-biotin or streptactin-biotin interactions and demonstrate its applicability for intracellular delivery of active proteins. We show that the nanogels can be loaded with proteins bearing either biotin, streptavidin, or strep-tag, and the resulting functionalized nanogels can be delivered into living cells after complexation with cationic lipid vectors. We use this approach for delivery of alkaline phosphatase enzyme, which is shown to keep its catalytic activity after internalization by mouse melanoma B16 cells, as demonstrated by the DDAO-phosphate assay. The resulting functionalized nanogels have dimensions on the order of 100 nm, contain around 100 enzyme molecules, and are shown to be transfectable at low lipid concentrations (charge ratio R± = 0.75). This ensures the low toxicity of our system, which in combination with high local enzyme concentration (∼100 μM) underlines potential interest of this nanoplatform for biomedical applications.

Zwitterionic materials for nucleic acid delivery and therapeutic applications

Nucleic acid therapeutics have demonstrated substantial potential in combating various diseases. However, challenges persist, particularly in the delivery of multifunctional nucleic acids. To address this issue, numerous gene delivery vectors have been developed to fully unlock the potential of gene therapy. The advancement of innovative materials with exceptional delivery properties is critical to propel the clinical translation of nucleic acid drugs. Cationic vector materials have received extensive attention, while zwitterionic materials remain relatively underappreciated in delivery. In this review, we outline a diverse range of zwitterionic material nucleic acid carriers, predominantly encompassing zwitterionic lipids, polymers and peptides. Their respective chemical structures, synthesis approaches, properties, advantages, and therapeutic applications are summarized and discussed. Furthermore, we highlight the challenges and future opportunities associated with the development of zwitterionic vector materials. This review will aid to understand the zwitterionic materials in aiding gene delivery, contributing to the continual progress of nucleic acid therapeutics.

Advancing breast cancer diagnosis with a near-infrared fluorescence imaging smart sensor for estrogen/progesterone receptor detection

Molecular-genetic imaging has greatly advanced clinical diagnosis and prognosis monitoring. However, the specific visualization of intracellular proteins such as estrogen receptor (ER) and progesterone receptor (PR) remains an elusive goal. Here, we highlight a novel method for selectively detecting ER/PR positive tumors using genetically engineered responsive elements. Our study demonstrates that the double responsive elements of ER/PR exhibit the most sensitivity to the steroid receptors in breast cancers. By utilizing a cationic polymer vector, we constructed a responsive element-fluorescence protein system that can selectively image ER/PR positive breast cancers in murine models under a near-infrared laser. This non-invasive imaging achieved high-resolution detection without death or serious anaphylactic activity in the animals. Our findings suggest that the reporter system consisting of steroid receptor response elements and near-infrared proteins provides a practical system for identifying biomarkers and advancing cancer diagnosis and therapy.

Cationic Materials for Gene Therapy: A Look Back to the Birth and Development of 2,2-Bis-(hydroxymethyl)Propanoic Acid-Based Dendrimer Scaffolds.

Gene therapy is extensively studied as a realistic and promising therapeutic approach for treating inherited and acquired diseases by repairing defective genes through introducing (transfection) the "healthy" genetic material in the diseased cells. To succeed, the proper DNA or RNA fragments need efficient vectors, and viruses are endowed with excellent transfection efficiency and have been extensively exploited. Due to several drawbacks related to their use, nonviral cationic materials, including lipidic, polymeric, and dendrimer vectors capable of electrostatically interacting with anionic phosphate groups of genetic material, represent appealing alternative options to viral carriers. Particularly, dendrimers are highly branched, nanosized synthetic polymers characterized by a globular structure, low polydispersity index, presence of internal cavities, and a large number of peripheral functional groups exploitable to bind cationic moieties. Dendrimers are successful in several biomedical applications and are currently extensively studied for nonviral gene delivery. Among dendrimers, those derived by 2,2-bis(hydroxymethyl)propanoic acid (b-HMPA), having, unlike PAMAMs, a neutral polyester-based scaffold, could be particularly good-looking due to their degradability in vivo. Here, an overview of gene therapy, its objectives and challenges, and the main cationic materials studied for transporting and delivering genetic materials have been reported. Subsequently, due to their high potential for application in vivo, we have focused on the biodegradable dendrimer scaffolds, telling the history of the birth and development of b-HMPA-derived dendrimers. Finally, thanks to a personal experience in the synthesis of b-HMPA-based dendrimers, our contribution to this field has been described. In particular, we have enriched this work by reporting about the b-HMPA-based derivatives peripherally functionalized with amino acids prepared by us in recent years, thus rendering this paper original and different from the existing reviews.

Reversible, Covalent DNA Condensation Approach Using Chemical Linkers for Enhanced Gene Delivery.

Nonviral gene delivery has emerged as a promising technology for gene therapy. Nonetheless, these approaches often face challenges, primarily associated with lower efficiency, which can be attributed to the inefficient transportation of DNA into the nucleus. Here, we report a two-stage condensation approach to achieve efficient nuclear transport of DNA. First, we utilize chemical linkers to cross-link DNA plasmids via a reversible covalent bond to form smaller-sized bundled DNA (b-DNA). Then, we package the b-DNA into cationic vectors to further condense b-DNA and enable efficient gene delivery to the nucleus. We demonstrate clear improvements in the gene transfection efficiency in vitro, including with 11.6 kbp plasmids and in primary cultured neurons. Moreover, we also observed a remarkable improvement in lung-selective gene transfection efficiency in vivo by this two-stage condensation approach following intravenous administration. This reversible covalent assembly strategy demonstrates substantial value of nonviral gene delivery for clinical therapeutic applications.

Elevating nucleic acid delivery via a stable anionic peptide-dextran ternary system.

Nucleic acid-based therapies hold promise for treating previously intractable diseases but require effective delivery vectors to protect the therapeutic agents and ensure efficient transfection. Cationic polymeric vectors are particularly notable for their adaptability, high transfection efficiency, and low cost, but their positive charge often attracts blood proteins, causing aggregation and reduced transfection efficiency. Addressing this, we designed an anionic peptide-grafted dextran (Dex-LipE5H) to serve as a cross-linkable coating to bolster the stability of cationic polymer/nucleic acid complexes. The Dex-LipE5H was synthesized through a Michael addition reaction, combining an anionic peptide (LipE5H) with dextran modified by divinyl sulfone. We demonstrated Dex-lipE5H utility in a novel ternary nucleic acid delivery system, CDex-LipE5H/PEI/nucleic acid. CDex-LipE5H/PEI/nucleic acid demonstrated lower cytotoxicity and superior anti-protein absorption ability compared to PEI/pDNA and Dex-LipE5H/PEI/pDNA. Most notably, the crosslinked CDex-LipE5H/PEI/pDNA demonstrated remarkable transfection performance in HepG2 cells, which poses significant transfection challenges, even in a medium with 20% serum. This system's effective siRNA interference performance was further validated through a PCSK9 gene knockdown assay. This investigation provides novel insights and contributes to the design of cost-effective, next-generation nucleic acid delivery systems with enhanced blood stability and transfection efficiency.

High-resolution bioenergetics correlates the length of continuous protonatable diaminoethane motif of four-armed oligo(ethanamino)amide transfectants to cytotoxicity

Recent clinical success with Onpattro and cationic ionizable lipid nanoparticle-based mRNA vaccines has rejuvenated research in the design and engineering of broader synthetic cationic vectors for nucleic acid compaction and transfection. However, perturbation of metabolic processes and cytotoxicity are still of concern with synthetic cationic vectors. Here, through an integrated bioenergetic and biomembrane integrity probing in three different human cell lines we reveal the dynamic effect of a library of sequence-defined four-arm oligo(ethanamino)amide transfectant on cell homeostasis, and identify metabolically safe building units over wide concentration ranges. The results show differential effects of the oligo(ethanamino)amide structure of comparable molecular weight on cell energetics. The severity of polycation effect on bioenergetic crisis follows with the length of continuous protonatable diaminoethane motif in the ascending order of glutaryl-triethylene tetramine, succinyl-tetraethylene pentamine and succinyl-pentaethylene hexamine. We further identify oligomeric structures that do not induce bioenergetic crisis even at high concentrations. Finally, transfection studies with a library of polyplexes carrying a reporter gene show no correlation between transfection efficiency and cytotoxicity. These observations demonstrate the usefulness of integrated high-resolution respirometry and plasma membrane integrity probing as a highly sensitive medium-throughput screening strategy for identification and selection of safe building units for transfectant engineering.

PEGylated pH-responsive peptide-mRNA nano self-assemblies enhance the pulmonary delivery efficiency and safety of aerosolized mRNA

Inhalable messenger RNA (mRNA) has demonstrated great potential in therapy and vaccine development to confront various lung diseases. However, few gene vectors could overcome the airway mucus and intracellular barriers for successful pulmonary mRNA delivery. Apart from the low pulmonary gene delivery efficiency, nonnegligible toxicity is another common problem that impedes the clinical application of many non-viral vectors. PEGylated cationic peptide-based mRNA delivery vector is a prospective approach to enhance the pulmonary delivery efficacy and safety of aerosolized mRNA by oral inhalation administration. In this study, different lengths of hydrophilic PEG chains were covalently linked to an amphiphilic, water-soluble pH-responsive peptide, and the peptide/mRNA nano self-assemblies were characterized by dynamic light scattering (DLS) and transmission electron microscopy (TEM). The in vitro mRNA binding and release, cellular uptake, transfection, and cytotoxicity were studied, and finally, a proper PEGylated peptide with enhanced pulmonary mRNA delivery efficiency and improved safety in mice was identified. These results showed that a proper N-terminus PEGylation strategy using 12-monomer linear monodisperse PEG could significantly improve the mRNA transfection efficiency and biocompatibility of the non-PEGylated cationic peptide carrier, while a longer PEG chain modification adversely decreased the cellular uptake and transfection on A549 and HepG2 cells, emphasizing the importance of a proper PEG chain length selection. Moreover, the optimized PEGylated peptide showed a significantly enhanced mRNA pulmonary delivery efficiency and ameliorated safety profiles over the non-PEGylated peptide and LipofectamineTM 2000 in mice. Our results reveal that the PEGylated peptide could be a promising mRNA delivery vector candidate for inhaled mRNA vaccines and therapeutic applications for the prevention and treatment of different respiratory diseases in the future.

A glutathione-sensitive cationic polymer delivery of CRISPR-Cas9 RNA plasmid for targeting nasopharyngeal carcinoma gene therapy

CRISPR-Cas9 technology has been proven to be the most straightforward and accurate tool for gene therapy, but some limitations, such as the inefficient transfection or inability to precisely target, prevent the gene therapy from achieving the desired therapeutic effect. To overcome these, a kind of glutathione-sensitive cationic vectors, hyperbranched polyamide amine (HPAA) was designed for Delivery of CRISPR-Cas9 RNA plasmid, and the cyclic RGD (Arg-Gly-Asp) was conjugated for the targeted treatment of nasopharyngeal carcinoma (NPC). Disulfide bonds in HPAA segments can specifically respond to the high glutathione concentration in the tumor microenvironment. Meanwhile, RGD could especially interact to the integrin αvβ3 receptors which are highly expressed on the surface of NPC tumor cells. The results showed that more HPAA-RGD/SGK3-gRNA complexes could be uptaken by NPC HNE-1 cells after RGD was conjugated, and then the plasmid could be accumulated in the NPC tumor as well, which may assure the satisfied NPC therapy effect in vivo. In transfection assays, this complex showed the acceptable gene transfection efficiency in vitro and the obvious tumor inhibition effect in vivo, suggested a potential application in gene therapy to NPC.

Fluorescent Self-Reporting Lipid Nanoparticles for Nitric Oxide/Gene Co-Delivery and Combination Therapy.

The combination cancer therapy of nitric oxide (NO) with gene therapy is a promising method for tumor treatment. However, efficient co-delivery of gas and therapeutic genes to tumor cells remains a challenge. Herein, we designed a nano-sized ultraviolet (UV) light-responsive cationic lipid vector DPNO(Zn). Fluorescence spectroscopy and confocal imaging experiments revealed that DPNO(Zn) lipid nanoparticles (LNPs) could rapidly release NO under low-power UV light irradiation. Moreover, the fluorescence turn-on might take place along with the release of NO, indicating the self-reporting ability. Gene delivery experiments showed that DPNO(Zn) LNPs had good gene transfection ability, making such materials a good candidate for gas/gene combination therapy. In vitro antitumor assay demonstrated that the co-delivery system was more effective in inhibiting tumor cell proliferation than individual NO or pTrail treatment. Studies on the mechanism of tumor cell apoptosis induced by NO/pTrail co-delivery showed that NO could not only effectively increase the accumulation of p53 protein in tumor cells, thereby promoting the activation of caspase-3, but also induce mitochondrial damage. On the other hand, the Trail protein expressed by pTrail gene could enhance the degree of NO-induced caspase-3 activation, indicating the synergistic effect. These results proved that DPNO(Zn) LNP may serve as a multifunctional nanocarrier for potential tumor therapy.

Plasmid DNA cationic non-viral vector complexes induce cytotoxicity-associated PD-L1 expression up-regulation in cancer cells in vitro

Non-viral vectors are promising nucleic acid carriers which have been utilized in gene-based cancer immunotherapy. The aim of this study is to compare the transfection efficiency and cytotoxicity of three cationic non-viral vectors namely Polyethylenimine (PEI), Lipofectamine 2000 (LPF) and stable nucleic acid lipid particles (SNALPs) of different lipid compositions, for the delivery of plasmid DNA (pDNA) expressing immunostimulatory molecules, OX40L or 4-1BBL, to cancer cells in vitro. The results indicate that PEI and LPF are efficient vectors for pDNA delivery with high transfection efficiency obtained. However, pDNA-PEI and pDNA-LPF complexes up-regulated the expression of programmed death ligand-1 (PD-L1) and induced significant cytotoxicity in both B16F10 and CT26 cell lines. The up-regulation of PD-L1 expression induced by pDNA-PEI and pDNA-LPF complexes was independent of cancer cell line, nor was it linked to the presence of GpC motifs in the pDNA. In contrast, the use of biocompatible SNALPs (MC3 and KC2 types) resulted in lower pDNA transfection efficiency, however no significant up-regulation of PD-L1 or cytotoxicity was observed. A strong correlation was found between up-regulation of PD-L1 expression and cytotoxicity. Up-regulation of PD-L1 expression could be mitigated with RNAi, maintaining expression at basal levels. Due to the improved biocompatibility and the absence of PD-L1 up-regulation, SNALPs represent a viable non-viral nucleic acid vector for delivery of pDNA encoding immunostimulatory molecules. The results of this study suggest that PD-L1 expression should be monitored when selecting commercial transfection reagents as pDNA vectors for cancer immunotherapy in vitro.

Hydrophilic Surface Modification of Cationic Unimolecular Bottlebrush Vectors Moderate pDNA and RNP Bottleplex Stability and Delivery Efficacy.

A cationic unimolecular bottlebrush polymer with chemically modified end-groups was synthesized to understand the impact of hydrophilicity on colloidal stability, nucleic acid delivery performance, and toxicity. The bottlebrush polymer template was synthesized using grafting-through techniques and was therefore composed of a polynorbornene backbone with poly(2-(dimethylamino)ethyl methacrylate) side chains with dodecyl trithiocarbonate end-groups. Postpolymerization modification was performed to fully remove the end-groups or install hydroxy and methoxy poly(ethylene glycol) functional groups on the bottlebrush exterior. The bottlebrush family was preformulated with biological payloads of pDNA and CRISPR-Cas9 RNP in both water and PBS to understand binding, aggregation kinetics, cytotoxicity, and delivery efficacy. Increasing end-group hydrophilicity and preformulation of bottleplexes in PBS increased colloidal stability and cellular viability; however, this did not always result in increased transfection efficiency. The bottlebrush family exemplifies how formulation conditions, polymer loading, and end-group functionality of bottlebrushes can be tuned to balance expression with cytotoxicity ratios and result in enhanced overall performance.

Effect of ultrasonic parameters on gene transfection efficiency and cell viability of the multifunctional microbubble in vitro

Studies have reported that microbubbles combined with focused ultrasound (FUS) can increase the membrane permeability of tumor cells by triggering membrane perforation (sonoporation) to improve brain tumor-targeted gene delivery. However, few studies focused on how to increase the efficiency of gene delivery to brain tumor cells with minor cell damage. The aim of this in vitro study was to find optimal parameters for efficient gene transfer into glioma cells using the VEGFR2-targeted cationic microbubbles gene vector (MB) for future brain tumor-targeted gene delivery. Transfection efficiency and cell viability were used to determine the optimal transfection parameters. Various MB concentrations (2 × 107 to 16 × 107/mL), post-transfection incubation times (1–3 days), acoustic pressures (0–1400 kPa), duty cycles (0.5–5%), pulse repetition frequencies (PRF) (0.5–2000 Hz) and cycle numbers (25–10000) were investigated. The results showed that the MB concentration of 4 × 107 MB/ml and the incubation time of 2 days were optimal conditions for maximum gene transfection efficiency and minimum cell damage. In addition, gene transfection efficiency and cell viability depended strongly on the acoustic exposure conditions. The optimal FUS parameters include an acoustic pressure of 700 kPa, a duty cycle of 5%, a PRF of 5Hz, and a cycle number of 10000. These findings support those MB concentrations and FUS parameters of the MB-mediated gene delivery system influence gene transfer efficiency and cell viability. They also describe our novel development of multifunctional MB for FUS-triggered gene delivery in brain tumor cells.

Design and Gene Delivery Application of Polymeric Materials in Cancer Immunotherapy

Immunotherapy has offered an alternative therapy method for cancer patients with metastatic tumors or who are not suitable for surgical resection. Different from traditional surgery, radiotherapy and chemotherapy, immunotherapy mainly restores the activity of the body’s own immune cells silenced in the tumor microenvironment to achieve anticancer therapy. Gene therapy which corrects abnormal expression of immune cells in tumor microenvironment by delivering exogenous genes to specific immune cells, is the most widely studied immunotherapy. Although most available gene delivery vectors are still viral vectors, the further application of viral vectors is still limited by the immunogenicity and mutagenesis. Based on this, cationic polymeric gene vectors with high flexibility, high feasibility, low cost and high safety have been widely used in gene delivery. The structural variability of polymers allows specific chemical modifications to be incorporated into polymer scaffolds to improve their physicochemical properties for more stable loading of genes or more targeted delivery to specific cells. In this review, we have summarized the structural characteristics and application potential in cancer immunotherapy of these polymeric gene vectors based on poly(L-lysine), poly(lactic-co-glycolic acid), polyethyleneimine, poly(amidoamine) and hydrogel system.

PH-sensitive packaging of cationic particles by an anionic block copolymer shell

Cationic non-viral vectors show great potential to introduce genetic material into cells, due to their ability to transport large amounts of genetic material and their high synthetic versatility. However, designing materials that are effective without showing toxic effects or undergoing non-specific interactions when applied systemically remains a challenge. The introduction of shielding polymers such as polyethylene glycol (PEG) can enhance biocompatibility and circulation time, however, often impairs transfection efficiency. Herein, a multicomponent polymer system is introduced, based on cationic and hydrophobic particles (P(nBMA46-co-MMA47-co-DMAEMA90), (PBMD)) with high delivery performance and a pH-responsive block copolymer (poly((N-acryloylmorpholine)-b-(2-(carboxy)ethyl acrylamide)) (P(NAM72-b-CEAm74), PNC)) as shielding system, with PNAM as alternative to PEG. The pH-sensitive polymer design promotes biocompatibility and excellent stability at extracellular conditions (pH 7.4) and also allows endosomal escape and thus high transfection efficiency under acidic conditions. PNC shielded particles are below 200 nm in diameter and showed stable pDNA complexation. Further, interaction with human erythrocytes at extracellular conditions (pH 7.4) was prevented, while acidic conditions (pH 6) enabled membrane leakage. The particles demonstrate transfection in adherent (HEK293T) as well as difficult-to-transfect suspension cells (K-562), with comparable or superior efficiency compared to commercial linear poly(ethylenimine) (LPEI). Besides, the toxicity of PNC-shielded particles was significantly minimized, in particular in K-562 cells and erythrocytes. In addition, a pilot in vivo experiment on bone marrow blood cells of mice that were injected with PNC-shielded particles, revealed slightly enhanced cell transfection in comparison to naked pDNA. This study demonstrates the applicability of cationic hydrophobic polymers for transfection of adherent and suspension cells in culture as well as in vivo by co-formulation with pH-responsive shielding polymers, without substantially compromising transfection performance.Graphical

Radicals Scavenging MOFs Enabling Targeting Delivery of siRNA for Rheumatoid Arthritis Therapy.

Macrophages play essential roles in the progression of rheumatoid arthritis (RA), which are polarized into the pro-inflammatory M1 phenotype with significant oxidative stress and cytokines excretion. Herein, an active targeting nanomedicine based on metal-organic frameworks (MOFs) to re-educate the diseased macrophages for RA therapy is reported. The MOFs are prepared via coordination between tannic acid (TA) and Fe3+ , and anti-TNF-α siRNA is loaded via a simple sonication process, achieving high loading capacity comparable to cationic vectors. The MOFs show excellent biocompatibility, and enable rapid endo/lysosome escape of siRNA via the proton-sponge effect for effective cytokines down-regulation. Importantly, such nanomedicine displays intrinsic radicals scavenging capability to eliminate a broad spectrum of reactive oxygen and nitrogen species (RONS), which in turn repolarizes the M1 macrophages into anti-inflammatory M2 phenotypes for enhanced RA therapy in combination with siRNA. The MOFs are further modified with bovine serum albumin (BSA) to allow cascade RA joint and diseased macrophages targeted delivery. As a result, an excellent anti-RA efficacy is achieved in a collagen-induced arthritis mice model. This work provides a robust gene vector with great translational potential, and offers a vivid example of rationally designing MOF structure with multifunctionalities to synergize with its payload for enhanced disease treatment.