The Interaction Mechanism of Intramuscular Gene Delivery Materials with Cell Membranes.

It has been confirmed that skeletal muscle cells have the capability to receive foreign plasmid DNA (pDNA) and express functional proteins. This provides a promisingly applicable strategy for safe, convenient, and economical gene therapy. However, intramuscular pDNA delivery efficiency was not high enough for most therapeutic purposes. Some non-viral biomaterials, especially several amphiphilic triblock copolymers, have been shown to significantly improve intramuscular gene delivery efficiency, but the detailed process and mechanism are still not well understood. In this study, the molecular dynamics simulation method was applied to investigate the structure and energy changes of the material molecules, the cell membrane, and the DNA molecules at the atomic and molecular levels. From the results, the interaction process and mechanism of the material molecules with the cell membrane were revealed, and more importantly, the simulation results almost completely matched the previous experimental results. This study may help us design and optimize better intramuscular gene delivery materials for clinical applications.

The Progress of Non-Viral Materials and Methods for Gene Delivery to Skeletal Muscle.

Since Jon A. Wolff found skeletal muscle cells being able to express foreign genes and Russell J. Mumper increased the gene transfection efficiency into the myocytes by adding polymers, skeletal muscles have become a potential gene delivery and expression target. Different methods have been developing to deliver transgene into skeletal muscles. Among them, viral vectors may achieve potent gene delivery efficiency. However, the potential for triggering biosafety risks limited their clinical applications. Therefore, non-viral biomaterial-mediated methods with reliable biocompatibility are promising tools for intramuscular gene delivery in situ. In recent years, a series of advanced non-viral gene delivery materials and related methods have been reported, such as polymers, liposomes, cell penetrating peptides, as well as physical delivery methods. In this review, we summarized the research progresses and challenges in non-viral intramuscular gene delivery materials and related methods, focusing on the achievements and future directions of polymers.

Can nanoparticles and nano‒protein interactions bring a bright future for insulin delivery?

Insulin therapy plays an essential role in the treatment of diabetes mellitus. However, frequent injections required to effectively control the glycemic levels lead to substantial inconvenience and low patient compliance. In order to improve insulin delivery, many efforts have been made, such as developing the nanoparticles (NPs)-based release systems and oral insulin. Although some improvements have been achieved, the ultimate results are still unsatisfying and none of insulin-loaded NPs systems have been approved for clinical use so far. Recently, nano‒protein interactions and protein corona formation have drawn much attention due to their negative influence on the in vivo fate of NPs systems. As the other side of a coin, such interactions can also be used for constructing advanced drug delivery systems. Herein, we aim to provide an insight into the advance and flaws of various NPs-based insulin delivery systems. Particularly, an interesting discussion on nano‒protein interactions and its potentials for developing novel insulin delivery systems is initiated.

The proper strategy to compress and protect plasmid DNA in the Pluronic L64-electropulse system for enhanced intramuscular gene delivery.

Intramuscular expression of functional proteins is a promising strategy for therapeutic purposes. Previously, we developed an intramuscular gene delivery method by combining Pluronic L64 and optimized electropulse, which is among the most efficient methods to date. However, plasmid DNAs (pDNAs) in this method were not compressed, making them unstable and inefficient in vivo. We considered that a proper compression of pDNAs by an appropriate material should facilitate gene expression in this L64-electropulse system. Here, we reported our finding of such a material, Epigallocatechin gallate (EGCG), a natural compound in green teas, which could compress and protect pDNAs and significantly increase intramuscular gene expression in the L64-electropulse system. Meanwhile, we found that polyethylenimine (PEI) could also slightly improve exogenous gene expression in the optimal procedure. By analysing the characteristic differences between EGCG and PEI, we concluded that negatively charged materials with strong affinity to nucleic acids and/or other properties suitable for gene delivery, such as EGCG, are better alternatives than cationic materials (like PEI) for muscle-based gene delivery. The results revealed that a critical principle for material/pDNA complex benefitting intramuscular gene delivery/expression is to keep the complex negatively charged. This proof-of-concept study displays the breakthrough in compressing pDNAs and provides a principle and strategy to develop more efficient intramuscular gene delivery systems for therapeutic applications.

Bionic cartilage acellular matrix microspheres as a scaffold for engineering cartilage.

Extracellular matrix (ECM) scaffolds made from decellularized natural cartilage have been successfully used in cartilage lesion repair, but allogeneic cartilage donors are always in shortage and xenogeneic cartilage tissues may have the risk of unknown disease transfer. In this study, we constructed artificial bionic cartilage microspheres by encapsulating MSCs in collagen microspheres and cultured in a chondrogenic-inducing medium. Then, acellular matrix microsphere (BCAMM) scaffolds were fabricated from the cultured microspheres at three different developmental stages. A novel technique was introduced to fabricate BCAMM scaffolds, which enabled the production and utilization of the scaffolds in a short time. Due to the differences in surface morphologies and biological compositions, the three BCAMM scaffolds showed different chondrogenic effects. The 10-day BCAMM (10-BCAMM) scaffold showed the best overall results, successfully inducing MSC chondrogenesis without any additional fetal bovine serum or induction components (TGF-ß or dexamethasone). In comparison, the 5-day BCAMM (5-BCAMM) scaffold showed potential osteogenic effects. The advantages of micron-sized BCAMMs are outlined, specifically in the easier decellularization process without grinding, homogeneous cell seeding and infiltration, chondrogenic induction and better fitting to the irregular lesion shape.

Tailoring the Supramolecular Structure of Guanidinylated Pullulan toward Enhanced Genetic Photodynamic Therapy.

In the progress of designing a gene carrier system, what is urgently needed is a balance of excellent safety and satisfactory efficiency. Herein, a straightforward and versatile synthesis of a cationic guanidine-decorated dendronized pullulan (OGG3P) for efficient genetic photodynamic therapy was proposed. OGG3P was able to block the mobility of DNA from a weight ratio of 2. However, G3P lacking guanidine residues could not block DNA migration until at a weight ratio of 15, revealing guanidination could facilitate DNA condensation via specific guanidinium-phosphate interactions. A zeta potential plateau (∼+23 mV) of OGG3P complexes indicated the nonionic hydrophilic hydroxyl groups in pullulan might neutralize the excessive detrimental cationic charges. There was no obvious cytotoxicity and hemolysis, but also enhancement of transfection efficiency with regard to OGG3P in comparison with that of native G3P in Hela and HEK293T cells. More importantly, we found that the uptake efficiency in Hela cells between OGG3P and G3P complexes was not markedly different. However, guanidination caused changes in uptake pathway and led to macropinocytosis pathway, which may be a crucial reason for improved transfection efficiency. After introducing a therapeutic pKillerRed-mem plasmid, OGG3P complexes achieved significantly enhanced KillerRed protein expression and ROS production under irradiation. ROS-induced cancer cells proliferation suppression was also confirmed. This study highlights the guanidine-decorated dendronized pullulan could emerge as a reliable nonviral gene carrier to specifically deliver therapeutic genes.

Tailoring the supramolecular structure of amphiphilic glycopolypeptide analogue toward liver targeted drug delivery systems.

Amphiphilic glycopolypeptide analogues have harboured great importance in the development of targeted drug delivery systems. In this study, lactosylated pullulan-graft-arginine dendrons (LP-g-G3P) was synthesized using Huisgen azide-alkyne 1,3-dipolar cycloaddition between lactosylated pullulan and generation 3 arginine dendrons bearing Pbf and Boc groups on the periphery. Hydrophilic lactosylated pullulan was selected for amphiphilic modification, aiming at specific lectin recognition. Macromolecular structure of LP-g-G3P combined alkyl, aromatic, and peptide dendritic hydrophobic moieties and was able to self-assemble spontaneously into core-shell nanoarchitectures with small particle sizes and low polydispersity in the aqueous media, which was confirmed by CAC, DLS and TEM. Furthermore, the polyaromatic anticancer drug (doxorubicin, DOX) was selectively encapsulated in the hydrophobic core through multiple interactions with the dendrons, including π-π interactions, hydrogen bonding and hydrophobic interactions. Such multiple interactions had the merits of enhanced drug loading capacity (16.89±2.41%), good stability against dilution, and excellent sustained release property. The cell viability assay presented that LP-g-G3P nanoparticles had an excellent biocompatibility both in the normal and tumor cells. Moreover, LP-g-G3P/DOX nanoparticles could be effectively internalized into the hepatoma carcinoma cells and dramatically inhibited cell proliferation. Thus, this approach paves the way to develop amphiphilic and biofunctional glycopolypeptide-based drug delivery systems.

Multi-Responsive "Turn-On" Nanocarriers for Efficient Site-Specific Gene Delivery In Vitro and In Vivo.

Systemic gene delivery is a complicated and multistep process that confronts numerous biological barriers. It remains a formidable challenge to exploit a single gene carrier with multiple features to combat all obstacles collectively. Herein, a multi-responsive "turn-on" polyelectrolyte complex (DNA/OEI-SSx /HA-SS-COOH, DSS) delivery system is demonstrated with a sequential self-assembly of disulfide-conjugated oligoethylenimine (OEI-SSx ) and disulfide bond-modified hyaluronic acid envelope (HA-SS-COOH) that can combat multiple biological barriers collectively when administered intravenously. DSS is designed to effectively accumulate at the tumor tissue and to be internalized into tumor cells by recognizing CD44. The multi-responsive "turn-on" DSS can respond to the alterations of hyaluronidases and glutathione at both the tumor site and at the intracellular milieu. Sequential degradation and detachment of the HA-SS-COOH envelope followed by the dissociation of the OEI-SSx/DNA inner core contributes to the activation of the endosomal escape and gene release functions, thus greatly enhancing nuclear gene delivery. A systematic investigation of DSS has revealed that the tumor accumulation ability, internalization, and endosome escape of the DSS nanocarriers, DNA unpacking and nuclear transportation are all remarkably improved by the multi-responsive "turn-on" design resulting in highly efficient gene transfection in vitro and in vivo.

Synthesis of Electroneutralized Amphiphilic Copolymers with Peptide Dendrons for Intramuscular Gene Delivery.

Intramuscular gene delivery materials are of great importance in plasmid-based gene therapy system, but there is limited information so far on how to design and synthesize them. A previous study showed that the peptide dendron-based triblock copolymer with its components arranged in a reversed biomembrane architecture could significantly increase intramuscular gene delivery and expression. Herein, we wonder whether copolymers with biomembrane-mimicking arrangement may have similar function on intramuscular gene delivery. Meanwhile, it is of great significance to uncover the influence of electric charge and molecular structure on the function of the copolymers. To address the issues, amphiphilic triblock copolymers arranged in hydrophilic-hydrophobic-hydrophilic structure were constructed despite the paradoxical characteristics and difficulties in synthesizing such hydrophilic but electroneutral molecules. The as-prepared two copolymers, dendronG2(l-lysine-OH)-poly propylene glycol2k(PPG2k)-dendronG2(l-lysine-OH) (rL2PL2) and dendronG3(l-lysine-OH)-PPG2k-dendronG3(l-lysine-OH) (rL3PL3), were in similar structure but had different hydrophilic components and surface charges, thus leading to different capabilities in gene delivery and expression in skeletal muscle. rL2PL2 was more efficient than Pluronic L64 and rL3PL3 when mediating luciferase, ß-galactosidase, and fluorescent protein expressions. Furthermore, rL2PL2-mediated growth-hormone-releasing hormone expression could significantly induce mouse body weight increase in the first 21 days after injection. In addition, both rL2PL2 and rL3PL3 showed good in vivo biosafety in local and systemic administration. Altogether, rL2PL2-mediated gene expression in skeletal muscle exhibited applicable potential for gene therapy. The study revealed that the molecular structure and electric charge were critical factors governing the function of the copolymers for intramuscular gene delivery. It can be concluded that, combined with the previous study, both structural arrangements either reverse or similar to the biomembrane are effective in designing such copolymers. It also provides an innovative way in designing and synthesizing new electroneutralized triblock copolymers, which could be used safely and efficiently for intramuscular gene delivery.

Self-assembly of pH-sensitive fluorinated peptide dendron functionalized dextran nanoparticles for on-demand intracellular drug delivery.

In this study, the amphiphilic fluorinated peptide dendrons functionalized dextran (FPD-HZN-Dex) via an acid-sensitive hydrazone linkage was successfully designed and prepared for the first time. We demonstrated a spontaneous self-assembly of amphiphilic FPD-HZN-Dex into the well-defined nanoparticles with the core-shell architecture in aqueous media, which is attributed to the efficient amphiphilic functionalization of dextran by the hydrophobic fluorinated peptide dendrons. The spherical morphology, uniform particle size and good storage stability of the prepared FPD-HZN-Dex nanoparticles were characterized by dynamic light scattering and transmission electron microscopy, respectively. In vitro drug release studies showed a controlled and pH dependent hydrophobic drug release profile. The cell viability assays show excellent biocompatibility of the FPD-HZN-Dex nanoparticles for both normal cells and tumor cells. Moreover, the FPD-HZN-Dex self-assembled systems based on pH-sensitive hydrazone linkage also can serve as stimulus bioresponsive carriers for on-demand intracellular drug delivery. These self-assembled nanoparticles exhibit a stimulus-induced response to endo/lysosome pH (pH 5.0) that causes their disassembly over time, enabling controlled release of encapsulated DOX. This work has unveiled a unique non-covalent interaction useful for engineering amphiphilic dendrons or dendrimers self-assembled systems.

The cytocompatability of polyhydroxyalkanoates coated with a fusion protein of PHA repressor protein (PhaR) and Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) polypeptide.

Microbial polyhydroxyalkanoates (PHAs) are a family of polyesters with biodegradability, biocompatibility and adjustable mechanical properties that are under intensive development for bioimplant applications. In this research, a fusion protein of PHA repressor protein (PhaR) and Lys-Gln-Ala-Gly-Asp-Val (KQAGDV) oligopeptide (PhaR-KQAGDV) was utilized to enhance the PHA cytocompatability via a mechanism of PhaR hydrophobically binding to PHA coupled with KQAGDV oligopeptide, a specific ligand to the integrins on the cell surface, for promotion of cell adhesion. The PhaR-KQAGDV fusion protein successfully produced and purified from recombinant E. coli was used to coat the surfaces of several PHA including poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx), respectively. The PhaR was observed to bind efficiently on all PHA surfaces measured by the fluorescence intensity of PhaR-EGFP as compared to the uncoated (PhaR negative) PHA films. The PHA surface hydrophilicity measured by water contact angles was significantly improved after PhaR-KQAGDV coating. Observations under confocal microscope and scanning electron microscopy, together with CCK-8 assays clearly demonstrated that adhesion and proliferation of human vascular smooth muscle cells (HvSMCs) inoculated on PHA films were much better on PhaR-KQAGDV coated surfaces than the non-coated control ones. The convenient physical coating approach for enhanced PHA cytocompatibility provides an advantage for PHA based tissue engineering.