Efficient Gene Carriers Research Articles

Polyethylenimine-modified graphene quantum dots promote endothelial cell proliferation.

Endothelial cell proliferation plays an important role in angiogenesis and treatment of related diseases. The aim of this study was to evaluate the effect of polyethylenimine (PEI)-modified graphene quantum dots (GQDs) gene vectors on endothelial cell proliferation. The GQDs-cationic polymer gene vectors were synthesized by amidation reaction, and used to deliver pZNF580 gene to Human umbilical vein endothelial cells (HUVECs) for promoting their proliferation. The chemical modification of GQDs can adjust gene vectors' surface properties and charge distribution, thereby enhancing their interaction with gene molecules, which could effectively compress the pZNF580 gene. The CCK-8 assay showed that the cell viability was higher than 80% at higher vector concentration (40 μg/mL), demonstrating that the GQDs-cationic polymer gene vectors and their gene complex nanoparticles (NPs) having low cytotoxicity. The results of the live/dead cell double staining assay were consistent with those of the CCK-8 assay, in which the cell viability of the A-GQDs/pZNF580 (94.38 ± 6.39%), C-GQDs-PEI- polylactic acid-co-polyacetic acid (PLGA)/pZNF580 (98.65 ± 6.60%) and N-GQDs-PEI-PLGA/pZNF580 (90.08 ± 1.60%) groups was significantly higher than that of the Lipofectamine 2000/pZNF580 (71.98 ± 3.53%) positive treatment group. The results of transfection and western blot experiments showed that the vector significantly enhanced the delivery of plasmid to HUVECs and increased the expression of pZNF580 in HUVECs. In addition, the gene NPs better promote endothelial cell migration and proliferation. The cell migration rate and proliferation ability of C-GQDs-PEI-PLGA/pZNF580 and N-GQDs-PEI-PLGA/pZNF580 treatment groups were higher than those of Lipofectamine 2000/pDNA treatment group. Modified GQDs possess the potential to serve as efficient gene carriers. They tightly bind gene molecules through charge and other non-covalent interactions, significantly improving the efficiency of gene delivery and ensuring the smooth release of genes within the cell. This innovative strategy provides a powerful means to promote endothelial cell proliferation.

Regulating the Obesity-Related Tumor Microenvironment to Improve Cancer Immunotherapy.

Obesity usually induces systemic metabolic disturbances, including in the tumor microenvironment (TME). This is because adaptive metabolism related to obesity in the TME with a low level of prolyl hydroxylase-3 (PHD3) depletes the major fatty acid fuels of CD8+ T cells and leads to the poor infiltration and unsatisfactory function of CD8+ T cells. Herein, we discovered that obesity could aggravate the immunosuppressive TME and weaken CD8+ T cell-mediated tumor cell killing. We have thus developed gene therapy to relieve the obesity-related TME to promote cancer immunotherapy. An efficient gene carrier was prepared by modifying polyethylenimine with p-methylbenzenesulfonyl (abbreviated as PEI-Tos) together with hyaluronic acid (HA) shielding, achieving excellent gene transfection in tumors after intravenous administration. HA/PEI-Tos/pDNA (HPD) containing the plasmid encoding PHD3 (pPHD3) can effectively upregulate the expression of PHD3 in tumor tissues, revising the immunosuppressive TME and significantly increasing the infiltration of CD8+ T cells, thereby improving the responsiveness of immune checkpoint antibody-mediated immunotherapy. Efficient therapeutic efficacy was achieved using HPD together with αPD-1 in colorectal tumor and melanoma-bearing obese mice. This work provides an effective strategy to improve immunotherapy of tumors in obese mice, which may provide a useful reference for the immunotherapy of obesity-related cancer in the clinic.

Superior gene transfection efficiency in triple negative breast cancer by RAFT-mediated amino acid-based cationic diblock copolymers.

To date, the synthesis of efficient and safe gene carriers with low toxicity and appreciable gene transfection efficiency has been the major hurdle associated with non-viral gene carriers. Herein, we synthesized three amino acid-based diblock copolymers comprising glycine-leucine, leucine-phenyl alanine and glycine-phenyl alanine group containing blocks. The synthesis of all the diblock copolymers was confirmed by FTIR, 1H NMR, DLS and GPC techniques. All the polymers showed a high positive zeta potential value that varies from 45 ± 1 mV to 56 ± 1 mV, and the hydrodynamic size of the polymers varies from 250 ± 8 to 303 ± 14 nm. The three polymers showed negligible cytotoxicity compared with PEI (25 kDa) for MDA-MB-231 and NKE cells. Among all other polymers, P(HGN)n-b-P(HPN)m exhibited the highest biocompatibility with ∼70% cell viability at a concentration of 200 μg mL-1. Hemolysis data revealed that among all three polymers, P(HGN)n-b-P(HPN)m exhibited the highest blood compatibility, while up to a high concentration of 200 μg mL-1, it showed a very negligible amount (∼18%) of hemolysis. Most importantly, excellent gene complexation capability and good protection of pDNA against enzymatic degradation were observed with all three diblock copolymers. Interestingly, P(HGN)n-b-P(HPN)m/pDNA complex showed the smallest particle size (∼15 nm) and highest positive zeta potential as observed from TEM micrographs and DLS analysis, which probably results significantly higher level of cellular uptake and hence the highest transfection efficiency (∼85%) against MDA-MB-231 cells. Therefore, the diblock copolymer P(HGN)n-b-P(HPN)m with superior gene transfection efficiency in triple negative breast cancer may be an efficient non-viral vector for successful TNBC therapy in the future.

Preparation of PEI-modified nanoparticles by dopamine self-polymerization for efficient DNA delivery.

Achieving efficient and safe gene delivery is of greatsignificance to promote the development of gene therapy. In this work, a polydopamine (PDA) layer was coated on the surface of Fe3 O4 nanoparticles (NPs) by dopamine (DA) self-polymerization, and then magnetic Fe3 O4 NPs were prepared by the Michael addition between amino groups in polyethyleneimine (PEI) and PDA. The prepared Fe3 O4 NPs (named Fe3 O4 @PDA@PEI) were characterized by Fourier transform infrared (FTIR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). As an efficient and safe gene carrier, the potential of Fe3 O4 @PDA@PEI was evaluated by agarose gel electrophoresis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, fluorescence microscopy, and flow cytometry. The results show that the Fe3 O4 @PDA@PEI NPs are stable hydrophilic NPs with a particle size of 50-150nm. It can efficiently condense DNA at low N/P ratios and protect it from nuclease degradation. In addition, the Fe3 O4 @PDA@PEI NPs have higher safety than PEI. Further, the Fe3 O4 @PDA@PEI/DNA polyplexes could be effectively absorbed by cells and successfully transfected and exhibit higher cellular uptake and gene transfection efficiency than PEI/DNA polyplexes. The findings indicate that the Fe3 O4 @PDA@PEI NPs have the potential to be developed into a novel gene vector.

Serum Compatible Spermine-Based Cationic Lipids with Nonidentical Hydrocarbon Tails Mediate High Transfection Efficiency.

Cationic lipids are widely used as nonviral synthetic vectors for gene delivery as a safer alternative to viral vectors. In this work, a library of L-shaped spermine-based cationic lipids with identical and nonidentical hydrophobic chains having variable carbon lengths (from C10 to C18) was designed and synthesized. These lipids were characterized and the structure-activity relationships of these compounds were determined for DNA binding and transfection ability when formulated as cationic liposomes. The liposomes were then used successfully for the transfection of HEK293T, HeLa, PC3, H460, HepG2, SH-SY5Y and Calu'3 cell lines. The transfection efficiency of lipids with nonidentical hydrocarbon chains was greater than the identical analogue. These reagents exhibited superior efficiency to the commercial reagent, Lipofectamine3000, under both serum-free and 10-40 % serum conditions in HEK293T, HeLa and H460 cell lines. The lipids were not toxic to the tested cell line. The results suggest that L-shaped spermine-based cationic lipids with nonidentical hydrocarbon tails could serve as efficient and safe nonviral vector gene carriers in further in vivo studies.

ROS-responsive fluorinated polyethyleneimine vector to co-deliver shMTHFD2 and shGPX4 plasmids induces ferroptosis and apoptosis for cancer therapy

Ferroptosis is a newly discovered non-apoptotic cell death form but its therapeutic efficacy triggered by traditional iron-based nanomaterials or classic drug inducers has been far from satisfactory due to the high glutathione (GSH) level in cancer cells and insufficient lipid peroxide production. Here we reported a ferroptosis/apoptosis combinational therapy by depleting GSH and downregulating GPX4 to disrupt redox homeostasis and amplify ferroptosis-related oxidation effect. In this study, we developed reactive oxygen species (ROS)-responsive serum-resistant nanoparticles with thioketal-crosslinked fluorinated polyethyleneimine 1.8K (TKPF) as the core, which were wrapped with hyaluronic acid (HA) as the shell (TKPFH NP) to co-deliver shGPX4 and shMTHFD2 plasmids for cancer treatment. The highly efficient and tumor-selective gene carrier TKPFH NPs revealed outstanding transfection efficiency (∼100 %) and sustained the efficiency (∼50 %) even in media containing 90 % FBS. Mediated by HA, TKPFH NPs actively targeted CD44 receptors, thus enabling efficient uptake by tumor cells and experiencing surface charge conversion to induce subsequent lysosomal escape. Then the TKPF NPs were effectively disintegrated by the abundant ROS in cancer cells, which facilitated the release of plasmids and avoided the cytotoxicity of cationic polymers. shGPX4 plasmid induced ferroptosis by producing ROS and lipid peroxides via downregulating GPX4, while shMTHFD2 triggered apoptosis by modulating NADPH/NADP and depleting GSH of the cancer cells. Moreover, GSH consumption caused by shMTHFD2 indirectly suppressed GPX4 and further augmented ferroptosis, showing synergistic anticancer effect against B16-F10 cells. Taken together, the rationally designed dual-gene loaded TKPFH NPs provided a safe and high-performance platform for enhanced ferroptosis-apoptosis combined anticancer efficacy based on gene therapy. Statement of significanceThe therapeutic efficacy of ferroptosis has been far from satisfactory due to high GSH level and insufficient lipid peroxide production in cancer cells. Herein, we reported a ferroptosis/apoptosis combinational therapy by depleting GSH and downregulating GPX4 to disrupt redox homeostasis and amplify ferroptosis-related oxidation effect. ROS-responsive serum-resistant nanoparticles were fabricated with thioketal-crosslinked fluorinated PEI 1.8K (TKPF) as the core and hyaluronic acid (HA) as the shell (TKPFH NP) to co-deliver shGPX4 and shMTHFD2 plasmids. The shGPX4 plasmid induced ferroptosis by producing ROS and lipid peroxides via downregulating GPX4, while shMTHFD2 triggered apoptosis by modulating NADPH/NADP and depleting GSH. The rationally designed dual-gene loaded TKPFH NPs provided a safe and high-performance platform aimed for enhanced ferroptosis-apoptosis combined anticancer efficacy.

ATP-Responsive Multifunctional Supramolecular Polymer as a Nonviral Vector for Boosting Cholesterol Removal from Lipid-Laden Macrophages.

Specific delivery of NCEH1 plasmid is a promising approach to boost the cholesterol removal from lipid-laden macrophages for antiatherosclerosis. Polyethylenimine (PEI) is one of the most efficient gene carriers among nonviral vectors. However, the high transfection activity of PEI is always accompanied by profound cytotoxicity. To tackle the paradox between transfection efficiency and safety, we constructed a novel ATP-responsive multifunctional supramolecular polymer by cross-linking functionalized low-molecular-weight PEI via a boronic ester bond for NCEH1 plasmid delivery. The supramolecular polymer could condense NCEH1 plasmids to form stable nanosized polyplexes when the w/w ratios of the polymer and gene were higher than 2. ATP-triggered degradation of the polymer and pDNA release were characterized by a series of studies, including 1H NMR, 31P NMR, XPS, agarose gel electrophoresis, and ethidium bromide exclusion tests. In addition, the changes in particle size and morphology were observed in the presence of ATP. Interestingly, the supramolecular polymer showed broad spectrum antioxidant activities by measuring the elimination rates of different reactive oxygen species. In addition, the supramolecular polymer displayed a high buffering capability and good cytocompatibility as demonstrated by the results of the buffering capacity, a hemolysis assay, and a cytotoxicity test. Importantly, it was revealed that the supramolecular polymer/NCEH1 plasmid polyplex formulated at a w/w ratio of 20 was most effective in enhancing cholesterol removal from lipid-laden macrophages and reducing the accumulation of lipid droplets as evidenced by transfection study, cholesterol efflux assay, and oil red O staining studies. Collectively, the ATP-responsive multifunctional supramolecular polymer holds great potential for safe and efficient gene delivery for antiatherosclerosis.

Synergistic gene delivery by self-assembled nanocomplexes using fusion peptide and calcium phosphate

Gene therapy can be a promising therapeutic approach to cure the fundamental causes of incurable genetic diseases. Because virus carriers are costly and can cause inflammation and immunogenicity, efficient non-viral carriers need to be developed for broader gene therapy applications. Therefore, we designed novel synergistic nanocomplexes for efficient transfection incorporated by the fusion of nuclear localization signal and cell-penetrating peptides with calcium phosphate. Fusion peptides were able to package large plasmid DNAs into nanocomplexes spontaneously and efficiently. After optimization, S-R/CaP or S-S/CaP nanocomplexes significantly improved specific luciferase expression up to 2-fold compared to Lipofectamine® 2000. In addition, the large Cas9-encoding plasmids were transfected into HEK293T cells more efficiently than Lipofectamine® 2000. Furthermore, subcutaneously injected cells to mice maintained more stable protein expression until 10 days than Lipofectamine® 2000. Moreover, the biocompatibility was revealed by observing negligible cytotoxicity, histological difference, and inflammatory cytokine release. Consequently, the new chimeric strategy will be an efficient and safe gene carrier into cells and tissues to treat various genetic diseases through gene therapy.

Design of Polymers for Intracellular Protein and Peptide Delivery

AbstractCytosolic protein delivery techniques are of great importance for cell biology, biotechnology and protein drug development. The design of carriers with robust efficiency in cytosolic protein delivery is challenging. This account provides a progress report of polymeric carriers for this purpose in our group. During the past years, we have developed several types of functionalized polymers for cytosolic protein and peptide delivery by engineering polymers with ligands such as guanidinium, boronate, coordination ligands and fluoroalkyls. The designed polymers showed improved protein/peptide binding affinities, and successfully delivered various cargo proteins into the cytosol of living cells, while maintaining their bioactivity. In addition, the polymers showed potent efficiencies in the delivery of tumor antigens, therapeutic peptides, toxins and antioxidant proteins in vivo. We hope these polymers could be translated for protein delivery in the treatment of various diseases in the future. What is the most favorite and original chemistry developed in your research group?Fluorinated polymers for cytosolic biomolecule delivery.How do you get into this specific field? Could you please share some experiences with our readers?I get into this field by unexpected discoveries when designing polymeric gene delivery systems. Usually, modification of polymers with hydrophobic ligands yields more efficient gene carriers. Fluoroalkyl ligands wereproposed as one type of hydrophobic candidates in our experiments. Interestingly, the fluorous ligand modified polymers showed unprecedented efficiency in gene delivery. More importantly, the polymers exhibited excellent serum resistance and could achieve high efficiency at extremely low polymer dose. These unexpected discoveries motivated us to investigate the fluorine effect of fluorinated materials in gene, protein and peptide delivery.

Fluoropolymer‐Mediated Intracellular Delivery of miR‐23b for the Osteocyte Differentiation in Osteoblasts

Emerging evidence suggests that microRNAs (miRNAs) play key roles in the regulation of multiple biological processes, including the differentiation of osteoblasts. Although miRNA-based gene therapy holds immense potential in the treatment of a variety of diseases, the intracellular delivery of miRNA remains challenging owing to the lack of efficient and safe gene carriers. In this study, a fluoropolymer (FP) is constructed through the modification of polyamidoamine (PAMAM) using heptafluorobutyric anhydride and then is used as a carrier for miR-23b transfection to induce osteocyte differentiation of osteoblasts. The derivative FP is found to facilitate miR-23b transfection due to its favorable endosomal escape from the "proton sponge" effect. Compared to PAMAM/miR-23b, the FP/miR-23b nanocomplex efficiently promotes the differentiation of osteoblasts and formation of calcified nodules, attributable to enhanced expression of various osteogenesis genes (runt-related transfection factor 2 [RUNX2], alkaline phosphatase [ALP], osteopontin [OPN], and osteocalcin [OCN]). Thus, FP-mediated miR-23b transfection may be used as an effective strategy to facilitate osteogenic differentiation.

ROS-Responsive Fluorinated Polyethyleneimine Vector to Co-Deliver shMTHFD2 and shGPX4 Plasmids Induces Ferroptosis and Apoptosis for Cancer Therapy

Ferroptosis is a novel cell death form against drug-resistance through bypassing apoptosis, but its therapeutic efficacy triggered by traditional iron-based nanomaterials or classic drug inducers has been far from satisfactory due to the high glutathione (GSH) level in tumor cells and insufficient lipid peroxide production. Here we reported a combinational ferroptosis/apoptosis therapy approach by depleting GSH and downregulating GPX4 to disrupt redox homeostasis and amplify ferroptosis-related oxidation effect. In this study, we developed a ROS-responsive serum-resistant nanoparticles with a thioketal-crosslinked fluorinated polyethyleneimine PEI 1.8K (TKPF) as the core and wrapped with hyaluronic acid (HA) as the shell (TKPFH NP) to co-deliver shGPX4 and shMTHFD2 plasmids for cancer treatment. The highly efficient and tumor-selective gene carrier TKPFH NPs revealed outstanding transfection efficiency (~100 %) and sustained the efficiency (~50 %) even in media containing 90 % FBS. TKPFH NPs actively targeted CD44 receptors mediated by HA to enable efficient uptake by tumor cells and experienced surface charge conversion to induce subsequently lysosomal escape. Then the TKPF NPs were effectively disintegrated by the abundant ROS in cancer cells, which facilitated release of plasmids and avoided the cytotoxicity of cationic polymers. shGPX4 plasmid induced ferroptosis by producing ROS and lipid peroxides via downregulating GPX4, while shMTHFD2 triggered apoptosis by modulating NADPH/NADP and depleting GSH of the cancer cells. Moreover, GSH consumption resulted by shMTHFD2 indirectly suppressed GPX4 and further augmented ferroptosis, showing synergistic anticancer effect against B16-F10 cells. Taken together, the rationally designed dual-gene loaded TKPFH NPs provided a safe and high-performance platform aimed for enhanced ferroptosis-apoptosis combined anticancer efficacy based on gene therapy.

N-Isopropylacrylamide-modified polyethylenimine-mediated miR-29a delivery to inhibit the proliferation and migration of lung cancer cells

MicroRNAs have been identified as a promising tool in cancer gene therapy, and an efficient and safe gene carrier was significantly required in the clinical application of miRNAs. Herein, a polyethylenimine (PEI) derivative, N-isopropylacrylamide-modified PEI (namely PEN), was constructed through Michael addition and then employed as a carrier for miR-29a transfection. The carrier PEN has been demonstrated to possess favorable ability to condense miR-29a into stable nanoparticles and protect miR-29a against the nuclease degradation, using agarose gel retardation assay. Meanwhile, PEN exhibited excellent efficiency in miR-29a transfection demonstrated by flow cytometry and confocal laser scanning microscope. Further, the PEN-mediated miR-29a transfection could achieve an obvious anti-proliferative effect owing to the activation of cell apoptosis and the cell cycle arrest at G1 phase, using human lung adenocarcinoma cell line A549 as a model. In addition, PEN/miR-29a nanoparticles could suppress the migration and invasion of cancer cells measured by wound healing and Transwell migration assays. Overall, the PEN-mediated miR-29a transfection could be potentially employed as a useful approach to achieve cancer gene therapy.

Brain gene delivery using histidine and arginine-modified dendrimers for ischemic stroke therapy

Polyamidoamine dendrimer has been studied as an efficient gene carrier. Due to its anti-inflammatory properties, polyamidoamine is a useful gene carrier, especially for inflammatory diseases. However, the commonly used polyamidoamine generation 6 dendrimer (PG6) has higher cytotoxicity than low-molecular weight polyamidoamines, which limits its applications. Therefore, early-generation polyamidoamine dendrimers, such as generation 2 (PG2), have been investigated as an alternative to PG6, although PG2 has a lower transfection efficiency. In this study, to improve gene delivery efficiency, histidine and arginine were conjugated on the primary amines of PG2, synthesizing PG2HR. The gene delivery efficiency of PG2HR was higher than that of PG2 or of PG2 conjugated with only arginine (PG2R), which may be due to higher cellular uptake and endosomal escape of the plasmid DNA (pDNA)/PG2HR complex. In addition, PG2HR had lower cytotoxicity than polyethylenimine (25 kDa, PEI25k), PG2, and PG2R. Mechanism studies showed that PG2HR delivered pDNA into the cells mainly by clathrin-independent endocytosis and partly by macropinocytosis. The therapeutic potential of PG2HR-mediated gene delivery was evaluated in middle cerebral artery occlusion (MCAO)-reperfusion stroke animal models. Heme oxygenase-1 (HO-1) plasmid was delivered into the brain by local injection. The results showed that PG2HR had higher gene delivery efficiency in the brain than did PEI25k, PG2, or PG2R. Furthermore, compared to the pHO-1/PEI25k, pHO-1/PG2, and pHO-1/PG2R complexes, the pHO-1/PG2HR complex had reduced apoptosis levels and infarct sizes in ischemic brains. Therefore, because of its low cytotoxicity and high gene delivery efficiency, PG2HR may be useful for gene therapy of inflammatory diseases including ischemic stroke.

Exosome as a Natural Gene Delivery Vector for Cancer Treatment.

Current gene therapy vectors such as viral, non-viral, and bacterial vectors, which are used for cancer treatment, but there are certain safety concerns and stability issues of these conventional vectors. Exosomes are the vesicles of size 40-100 nm secreted from multivesicular bodies into the extracellular environment by most of the cell types in-vivo and in-vitro. As a natural nanocarrier, exosomes are immunologically inert, biocompatible, and can cross biological barriers like the blood-brain barrier, intestinal barrier, and placental barrier. This review focusses on the role of exosome as a carrier to efficiently deliver a gene for cancer treatment and diagnosis. The methods for loading of nucleic acids onto the exosomes, advantages of exosomes as a smart intercellular shuttle for gene delivery and therapeutic applications as a gene delivery vector for siRNA, miRNA and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and also the limitations of exosomes as a gene carrier are all reviewed in this article. Mostly, electroporation and chemical transfection are used to prepare gene loaded exosomes. Exosome-mediated delivery is highly promising and advantageous in comparison to the current delivery methods for systemic gene therapy. Targeted exosomes, loaded with therapeutic nucleic acids, can efficiently promote the reduction of tumor proliferation without any adverse effects. In the near future, exosomes can become an efficient gene carrier for delivery and a biomarker for the diagnosis and treatment of cancer.

Dual-Location Dual-Acid/Glutathione-Degradable Cationic Micelleplexes through Hydrophobic Modification for Enhanced Gene Silencing.

Gene therapy holds great promise for the treatment of acquired genetic disorders such as cancer with reduced side effects compared to chemotherapy. For gene therapy to be successful, it is crucial to develop efficient and nontoxic gene carriers to overcome the poor in vivo stability and low cellular uptake of nucleic acid-based therapeutic agents. Here, we report a new and versatile approach exploring a combination of hydrophobic modifications and dual-stimuli-responsive degradation (SRD) for controlled gene delivery with amphiphilic block copolymer-based nanocarriers. The block copolymer, synthesized by atom transfer radical polymerization, is designed with an acid-labile acetal linkage at the block junction and a pendant disulfide group in the hydrophobic block. The incorporation of labile linkages enables both disulfide-core-cross-linking and dual-location dual-acid/reduction-responsive degradation (DL-DSRD). Furthermore, the disulfide linkages integrated as hydrophobic moieties facilitate the nucleic acids to condense into nanometer-sized micelleplexes through electrostatic interactions of pendant dimethylamino groups with the anionic phosphate groups of the nucleic acids. Our preliminary results demonstrate that the DL-DSRD approach through hydrophobic modification is a robust platform in the development of gene delivery systems with enhanced colloidal stability, reduced cytotoxicity, and improved gene transfection efficiency.

Dual-Modified Liposome for Targeted and Enhanced Gene Delivery into Mice Brain.

The development of neuropharmaceutical gene delivery systems requires strategies to obtain efficient and effective brain targeting as well as blood-brain barrier (BBB) permeability. A brain-targeted gene delivery system based on a transferrin (Tf) and cell-penetrating peptide (CPP) dual-functionalized liposome, CPP-Tf-liposome, was designed and investigated for crossing BBB and permeating into the brain. We selected three sequences of CPPs [melittin, Kaposi fibroblast growth factor (kFGF), and penetration accelerating sequence-R8] and compared their ability to internalize into the cells and, subsequently, improve the transfection efficiency. Study of intracellular uptake indicated that liposomal penetration into bEnd.3 cells, primary astrocytes, and primary neurons occurred through multiple endocytosis pathways and surface modification with Tf and CPP enhanced the transfection efficiency of the nanoparticles. A coculture in vitro BBB model reproducing the in vivo anatomophysiological complexity of the biologic barrier was developed to characterize the penetrating properties of these designed liposomes. The dual-functionalized liposomes effectively crossed the in vitro barrier model followed by transfecting primary neurons. Liposome tissue distribution in vivo indicated superior ability of kFGF-Tf-liposomes to overcome BBB and reach brain of the mice after single intravenous administration. These findings demonstrate the feasibility of using strategically designed liposomes by combining Tf receptor targeting with enhanced cell penetration as a potential brain gene delivery vector. SIGNIFICANCE STATEMENT: Rational synthesis of efficient brain-targeted gene carrier included modification of liposomes with a target-specific ligand, transferrin, and with cell-penetrating peptide to enhance cellular internalization. Our study used an in vitro triple coculture blood-brain barrier (BBB) model as a tool to characterize the permeability across BBB and functionality of designed liposomes prior to in vivo biodistribution studies. Our study demonstrated that rational design and characterization of BBB permeability are efficient strategies for development of brain-targeted gene carriers.

In Vitro Assessment of Core-Shell Micellar Nanostructures of Amphiphilic Cationic Polymer-Peptide Conjugates as Efficient Gene and Drug Carriers

Design and development of biocompatible, biodegradable and stable dual delivery systems for drug and gene is the need of the hour. Here, we have designed a strategy to develop carrier systems consisting of above mentioned properties by (a) incorporating an unnatural amino acid in the peptide backbone, and b) conjugating a low molecular weight cationic polymer (polyethylenimine, PEI) for incorporating cationic charge. Using this strategy, we have synthesized a small series of Boc-FΔF-AH-polyethylenimine conjugates by varying the concentration of Boc-FΔF-aminohexanoic acid, viz., PP-1, PP-2 and PP-3. These conjugates self-assembled in aqueous medium to form micelles in the size range of ~144–205 nm with zeta potential ~ +7.9–14.2 mV bearing core-shell type of conformation. Positive surface of the micelles facilitated the binding of plasmid DNA as well as transportation inside the cells. The hydrophobic core of the nanostructures helped in the encapsulation of the hydrophobic drug molecule, which was then got released in a controlled manner. DNA complexes of the conjugates were not only found non-toxic but also exhibited higher transfection efficacy than the native polymer and Lipofectamine. Altogether, these nanostructures are capable of delivering a drug and a gene simultaneously in vitro and could be used as next-generation delivery agents.

Cationic Heteropolymers with Various Functional Groups as Efficient and Biocompatible Nonviral Gene Vectors.

With the rise and development of gene therapy, it is of great significance to develop highly efficient and biocompatible polymeric gene carriers. In this work, a series of heteropolymers from ring-opening polymerization of diepoxide compounds and various functional primary amines were synthesized. The feed dosage of amines was adjusted to obtain the polymers with different functional group contents, and the structure-activity relationships of these polymers as nonviral gene vectors were examined in detail. Results revealed that, although the amine with the fluorinated chain seemed to be less reactive in the polymerization, the relative content of each component in the target product was consistent with the feed dosage. Compared to the "golden standard" polyethylenimine (PEI) 25 kDa, these heteropolymers showed much lower cytotoxicity and higher gene transfection efficiency, especially in serum-containing medium, and up to 78 times of efficiency than PEI was obtained. Meanwhile, they exhibited much better serum resistance than PEI, compared with the transfection efficiency in serum-free experiments, and even higher efficiency could be achieved with serum in HeLa cells. Mechanism study results suggest that the content of fluorinated chain and histamine might distinctly influence their transfection. The fluorinated chains could enhance the serum tolerance and cellular uptake efficiency (with serum), while the imidazole group in the histamine chain would improve the endosome/lysosome escape.

Highly efficient and tumor-selective nanoparticles for dual-targeted immunogene therapy against cancer.

While immunotherapy holds great promise for combating cancer, the limited efficacy due to an immunosuppressive tumor microenvironment and systemic toxicity hinder the broader application of cancer immunotherapy. Here, we report a combinatorial immunotherapy approach that uses a highly efficient and tumor-selective gene carrier to improve anticancer efficacy and circumvent the systemic toxicity. In this study, we engineered tumor-targeted lipid-dendrimer-calcium-phosphate (TT-LDCP) nanoparticles (NPs) with thymine-functionalized dendrimers that exhibit not only enhanced gene delivery capacity but also immune adjuvant properties by activating the stimulator of interferon genes (STING)-cGAS pathway. TT-LDCP NPs delivered siRNA against immune checkpoint ligand PD-L1 and immunostimulatory IL-2-encoding plasmid DNA to hepatocellular carcinoma (HCC), increased tumoral infiltration and activation of CD8+ T cells, augmented the efficacy of cancer vaccine immunotherapy, and suppressed HCC progression. Our work presents nanotechnology-enabled dual delivery of siRNA and plasmid DNA that selectively targets and reprograms the immunosuppressive tumor microenvironment to improve cancer immunotherapy.

Supramolecular Assembly of Amino Acid Based Cationic Polymer for Efficient Gene Transfection Efficiency in Triple Negative Breast Cancer.

The success of gene therapy is enormously dependent on an efficient gene carrier, and in this context, cationic polymers still continue to play a major role particularly with respect to the safety issue compared to viral vectors. Developing an efficient gene carrier system having promising gene transfection efficiency with low toxicity is the foremost impediment associated with a nonviral carrier. Here, we explored amino acid based biocompatible polymers synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization where glycine (Gly), leucine (Leu), and phenyl alanine (Phe) amino acids were used as the pendent groups of the polymeric brushes. The presence of both a hydrophobic group (long chain aliphatic group associated with the RAFT agent) and hydrophilic amino groups was associated with the supramolecular assembly of the polymeric chain having hydrodynamic sizes within the range of 150-300 nm with a positive zeta potential of 30 ± 5 mV. All polymers showed very low toxicity and possessed >80% cell viability even at a very high concentration of 1000 μg/mL against both normal and cancerous cells. In addition to this, the polymers also showed excellent blood compatibility, and negligible hemolysis was observed at the concentration of 500 μg/mL. All polymers showed efficient DNA complexation capability as well as excellent protection of DNA against highly negatively charged surfactant and enzymatic digestion, although the efficiency was dependent on the N/P ratio of polymer/DNA complexes. Interestingly, the phenyl alanine moiety containing polymer brush P(HEMA-Phe-NH2) showed a hexagonal shaped nanoparticle after complexation with pDNA and consequently showed higher cellular uptake, resulting in a higher transfection efficiency in a triple negative breast cancer cell, the MDA-MB-231 cell. Therefore, the synthesized polymer containing an amino acid pendent group, especially the phenyl alanine moiety, may be a promising nonviral gene carrier system in gene therapy application in the future.