Cuifang Cai

Influence of morphology and drug distribution on the release process of FITC-dextran-loaded microspheres prepared with different types of PLGA

The aim of the present work was to understand the collaborative roles and the comprehensive effects of polymer nature, morphology, drug distribution and release behaviour for PLGA microspheres prepared by the double emulsion method. The morphology and drug distribution of the FITC-dextran-loaded microspheres were investigated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), respectively. The results show that the morphology and release profiles depend on the polymer nature. For the capped PLGA RG502, the porosity, pore size and drug distribution had no pronounced influence on the release profile beyond the initial release. No significant changes in size and morphology were found and there was negligible water uptake during the release process. PEG addition as a pore maker indicated a possible way to modify the release rate at the second release stage. However, in the case of the uncapped PLGA RG503H, release profiles were dependent upon changes in porosity, pore size and drug loading. Increases in porosity, internal pore size and loading resulted in a continuous release profile. Previous studies have shown the importance of different process parameters on morphology and drug release, but in this work it is clear that polymer nature is a determining factor.

Guanidinylated bioresponsive poly(amido amine)s designed for intranuclear gene delivery

Guanidinylated poly(amido amine)s with multiple disulfide linkages (Gua-SS-PAAs) were designed and constructed as nonviral gene carriers. The main chains of these novel carriers were synthesized based on monomers containing guanidino groups (guanidine hydrochloride and chlorhexidine), which could avoid complicated side-chain-modification reactions while introducing the guanidino groups. The synthesized Gua-SS-PAAs polymers were characterized by 1H nuclear magnetic resonance, molecular weight, and polydispersity. Furthermore, Gua-SS-PAAs polymers were complexed with pDNA, and the properties of the complexes were determined, including entrapment efficiency, particle size, ζ-potential, atomic force microscopy images, stability, DNA complexation ability, reduction sensitivity, cytotoxicity, and transfection efficiency. The new Gua-SS-PAAs carriers exhibited higher transfection efficiency and lower cytotoxicity compared with two widely used gene delivery carriers, polyethylenimine and lipofectamine 2000. Furthermore, the relationship between the side-chain structure and morphological/biological properties was extrapolated, and the results showed that guanidine in the side chain aids in the improvement of transfection efficiency. In addition, the introduction of guanidino group might confer the new carriers with nuclear localization function compared to carriers without it.

Recent advances on extracellular vesicles in therapeutic delivery: Challenges, solutions, and opportunities

Graphical abstract Figure. No Caption available. Abstract Extracellular vesicles (EVs) are intrinsic mediators of intercellular communication in our body, allowing functional transfer of biomolecules (lipids, proteins, and nucleic acid) between diverse locations. Such an instrumental role evokes a surge of interest within the drug delivery community in tailoring EVs for therapeutic delivery. These vesicles represent a novel generation of drug delivery systems, providing high delivery efficiency, intrinsic targeting properties, and low immunogenicity. In the recent years, considerable research efforts have been directed toward developing safe and efficient EV‐based delivery vehicles. Although EVs are shown to harbor great promise in therapeutic delivery, substantial improvements in exploring standardized isolation techniques with high efficiency and robust yield, scalable production, standard procedures for EV storage, efficient loading methods without damaging EV integrity, understanding their in vivo trafficking, and developing novel EV‐based nanocarriers are still required before their clinical transformation. In this review, we seek to summarize the recent advance on harnessing EVs for drug delivery with focus on state‐of‐the‐art solutions for overcoming major challenges.

Novel guanidinylated bioresponsive poly(amidoamine)s designed for short hairpin RNA delivery

Two different disulfide (SS)-containing poly(amidoamine) (PAA) polymers were constructed using guanidino (Gua)-containing monomers (ie, arginine [Arg] and agmatine [Agm]) and N,N′-cystamine bisacrylamide (CBA) by Michael-addition polymerization. In order to characterize these two Gua-SS-PAA polymers and investigate their potentials as short hairpin RNA (shRNA)-delivery carriers, pSilencer 4.1-CMV FANCF shRNA was chosen as a model plasmid DNA to form complexes with these two polymers. The Gua-SS-PAAs and plasmid DNA complexes were determined with particle sizes less than 90 nm and positive ζ-potentials under 20 mV at nucleic acid:polymer weight ratios lower than 1:24. Bioresponsive release of plasmid DNA was observed from both newly constructed complexes. Significantly lower cytotoxicity was observed for both polymer complexes compared with polyethylenimine and Lipofectamine 2000, two widely used transfection reagents as reference carriers. Arg-CBA showed higher transfection efficiency and gene-silencing efficiency in MCF7 cells than Agm-CBA and the reference carriers. In addition, the cellular uptake of Arg-CBA in MCF7 cells was found to be higher and faster than Agm-CBA and the reference carriers. Similarly, plasmid DNA transport into the nucleus mediated by Arg-CBA was more than that by Agm-CBA and the reference carriers. The study suggested that guanidine and carboxyl introduced into Gua-SS-PAAs polymers resulted in a better nuclear localization effect, which played a key role in the observed enhancement of transfection efficiency and low cytotoxicity. Overall, two newly synthesized Gua-SS-PAAs polymers demonstrated great potential to be used as shRNA carriers for gene-therapy applications.

Structure–Function Correlations of Poly(Amido Amine)s for Gene Delivery

Poly(amido amine)s (PAAs) versatility are nearly unique among stepwise polymers. Different functional groups can be easily introduced into these polymers to add functionality such as cell internalization, charge-shift, bioreducibility, stealth properties, and targeting moieties, while maintaining the bulk structural integrity of these polymers. The poly(amido amine)s are used as a unique research platform to elucidate their complex structure-function relationship. It is shown that guanidinium group, carboxyl group, disulfide bond, alkyl chain, branching, acetyl groups, benzoyl groups, and quaternary nicotinamide moieties can influence many steps of gene delivery, such as DNA condensation, cellular uptake, endosomal escape, nuclear entry, and finally gene expression. The authors systematically discuss the structure-function correlations of PAAs for gene delivery, and elaborate how the properties of polymers can be adjusted by changing the polymeric structure.

Membrane-Loaded Doxorubicin Liposomes Based on Ion-Pairing Technology with High Drug Loading and pH-Responsive Property

In order to achieve high drug loading and high entrapment efficiency, a doxorubicin-cholesteryl hemisuccinate ion-pair complex (DCHIP) was formed, and the ion-pair complex liposomes (DCHIP-Lip) were prepared based on conventional thin-film dispersion method. Firstly, DCHIP was fabricated and confirmed with FTIR, 1H-NMR, DSC, and XRD techniques. Afterwards, DCHIP-Lip were prepared and evaluated in terms of particle size, zeta potential, entrapment efficiency, and drug loading content. Finally, the in vitro and in vivo behavior of liposomes was further investigated. The DCHIP-Lip had a nanoscale particle size of about 120xa0nm with a negative zeta potential of about −22xa0mV. In addition, the entrapment efficiency and drug loading content of DOX reached 6.4u2009±u20090.05 and 99.29u2009±u20090.3%, respectively. Importantly, the release of DCHIP-Lip was pH sensitive and increased cell toxicity against MCF-7 cells was achieved. Upon dilution, the liposomes were fairly stable under physiological conditions. The in vivo pharmacokinetic study indicated that the AUC of DOX in DCHIP-Lip was 11.48-fold higher than that of DOX-HCl solution and the in vivo antitumor activity of DCHIP-Lip showed less body weight loss and a significant prohibition effect of tumor growth. Based on these findings, it can be seen that the ion-pairing technology combined with conventional liposome drug loading method could be used to achieve high drug loading and it could be valuable for the study of liposomal delivery system.

Exploring the role of peptides in polymer-based gene delivery

Polymers are widely studied as non-viral gene vectors because of their strong DNA binding ability, capacity to carry large payload, flexibility of chemical modifications, low immunogenicity, and facile processes for manufacturing. However, high cytotoxicity and low transfection efficiency substantially restrict their application in clinical trials. Incorporating functional peptides is a promising approach to address these issues. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we systematically summarize the role of peptides in polymer-based gene delivery, and elaborate how to rationally design polymer-peptide based gene delivery vectors.nnnSTATEMENT OF SIGNIFICANCEnPolymers are widely studied as non-viral gene vectors, but suffer from high cytotoxicity and low transfection efficiency. Incorporating short, bioactive peptides into polymer-based gene delivery systems can address this issue. Peptides demonstrate various functions in polymer-based gene delivery systems, such as targeting to specific cells, breaching membrane barriers, facilitating DNA condensation and release, and lowering cytotoxicity. In this review, we highlight the peptides roles in polymer-based gene delivery, and elaborate how to utilize various functional peptides to enhance the transfection efficiency of polymers. The optimized peptide-polymer vectors should be able to alter their structures and functions according to biological microenvironments and utilize inherent intracellular pathways of cells, and consequently overcome the barriers during gene delivery to enhance transfection efficiency.

Disulfide-bond-containing agamatine-cystaminebisacrylamide polymer demonstrates better transfection efficiency and lower cytotoxicity than polyethylenimine in NIH/3T3 cells

Previously, we synthesized a non‐viral vector containing disulfide bond by polymerization of agamatine (AGM) and N,N′‐cystaminebisacrylamide (CBA). In this study, we investigated the transfection efficiency of disulfide bond (uf8ffSSuf8ff) containing AGM‐CBA polymer in gene delivery into NIH/3T3 cells, and examined the factors affecting its transfection efficiency by comparing with polyethylenimine (PEI). In addition, experiments were carried out to determine the mechanisms of cell entry pathways and intracellular behavior of AGM‐CBA/pDNA polyplexes. The transfection efficiency of AGM‐CBA/pDNA with different weight ratios and different amounts of pDNA was measured and the pathways mediated transfection processes were studied by using various endocytosis inhibitors. To determine the intracellular behavior of AGM‐CBA/pDNA polyplexes, the transfection efficiencies of AGM‐CBA/pDNA and PEI/pDNA polyplexes with different combination structures were determined by using reporter gene and fake plasmid DNA. The transfection efficiency of AGM‐CBA/pDNA polyplexes was correlated with its weight ratio of AGM‐CBA and pDNA, and the amount of pDNA. Both AGM‐CBA/pDNA and PEI/pDNA polyplexes enter into cell by clathrin‐ and caveolae‐mediated endocytic pathways. However, AGM‐CBA/pDNA showed different intracellular behavior in NIH/3T3 cells compared to PEI/pDNA polyplexes. It was hypothesized that disulfide bond in AGM‐CBA could be an important factor contributing to its intracellular behavior and better transfection efficiency. Overall, AGM‐CBA demonstrated better transfection efficiency and lower cytotoxicity than PEI in NIH/3T3 cells as a gene delivery vector.

Comparison of exosome-mimicking liposomes with conventional liposomes for intracellular delivery of siRNA

Graphical abstract Figure. No Caption available. Abstract Exosomes have been extensively explored as delivery vehicles due to low immunogenicity, efficient cargo delivery, and possibly intrinsic homing capacity. However, therapeutic application of exosomes is hampered by structural complexity and lack of efficient techniques for isolation and drug loading. Liposomes represent one of the most successful therapeutic nanocarriers, but are frequently criticized by short blood circulation and inefficient intracellular drug delivery. In this circumstance, a promising strategy is to facilitate a positive feedback between two fields. Herein, exosome‐mimicking liposomes were formulated with DOPC/SM/Chol/DOPS/DOPE (21/17.5/30/14/17.5, mol/mol), and harnessed for delivery of VEGF siRNA to A549 and HUVEC cells. Compared with Lipo 2000 and DOTAP liposomes, exosome‐mimicking liposomes exhibited less than four‐fold cytotoxicity but higher storage stability and anti‐serum aggregation effect. Exosome‐mimicking liposomes appeared to enter A549 cells through membrane fusion, caveolae‐mediated endocytosis, and macropinocytosis, while enter HUVEC through caveolae‐mediated endocytosis, which revealed that the uptake pathway was dependent on cell types. Notably, exosome‐mimicking liposomes exhibited significantly higher cellular uptake and silencing efficiency than PC‐Chol liposomes (>three‐fold), suggesting the unique lipid composition did enhance the intracellular delivery efficiency of exosome‐mimicking liposomes to a significantly greater extent. However, it still remained far from satisfactory delivery as compared to cationic Lipo 2000 and DOTAP liposomes, which warranted further improvement in future research. This study may encourage further pursuit of more exosome‐mimicking delivery vehicles with higher efficiency and biocompatibility.

Functionalized extracellular vesicles as advanced therapeutic nanodelivery systems

&NA; Extracellular vesicles (EVs) are membrane enclosed vesicles that are shed by almost all cell types, and play a fundamental role in cell‐to‐cell communication. The discovery that EVs are capable of functionally transporting nucleic acid‐ and protein‐based cargoes between cells, rapidly promotes the idea of employing them as drug delivery systems. These endogenous vesicles indeed hold tremendous promise for therapeutic delivery. However, issues associated with exogenously administered EVs, including rapid clearance by the immune system, apparent lack of targeting cell specificity, and insufficient cytoplasmic delivery efficiency, may limit their therapeutic applicability. In this review, we discuss recent research avenues in EV‐based therapeutic nanodelivery systems. Furthermore, we narrow our focus on the development of modification strategies to enhance the delivery properties of EVs, and elaborate on how to rationally harness these functionalized vesicles for therapeutic delivery. Graphical abstract Figure. No caption available.