Shen Gao

Second-generation aptamer-conjugated PSMA-targeted delivery system for prostate cancer therapy

Background: miR-15a and miR-16-1 have been identified as tumor suppressor genes in prostate cancer, but their safe and effective delivery to target cells is key to the successful use of this therapeutic strategy. RNA aptamer A10 has been used as a ligand, targeting prostate cancer cells that express prostate-specific membrane antigen (PSMA). Compared with A10, the binding of the second-generation RNA aptamer, A10-3.2, to PSMA is more efficient. Methods: A10-3.2 was investigated as a PSMA-targeting ligand in the design of a polyamidoamine (PAMAM)-based microRNA (miR-15a and miR-16-1) vector to prostate cancer cells. Using polyethyleneglycol (PEG) as a spacer, PAMAM was conjugated to aptamer (PAMAM-PEG-APT) and used as a vehicle for miRNA target delivery. Results: Luciferase assays of pGL-3 expression against PC3 (PSMA−) and LNCaP (PSMA+) cells demonstrated that the transfection efficiency of the synthesized DNA/PAMAM-PEG-APT complex was higher than that of the DNA/PAMAM-PEG complex. In addition, cell viability assays of LNCaP (PSMA+) cells showed that, with a N/P ratio of 15:1, the IC50 value of miRNA/PAMAM-PEG-APT was approximately 4.7-fold lower than that of miRNA/PAMAM-PEG. Conclusion: This PSMA-targeted system may prove useful in widening the therapeutic window and allow for selective killing of prostate cancer cells.

Penetration and distribution of PLGA nanoparticles in the human skin treated with microneedles.

This study was designed to investigate the penetration and the distribution of poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles in the human skin treated with microneedles. Fluorescent nanoparticles were prepared to indicate the transdermal transport process of the nanoparticles. Permeation study was performed on Franz-type diffusion cells in vitro. The distribution of nanoparticles was visualized by confocal laser scanning microscopy (CLSM) and quantified by high performance liquid chromatography (HPLC). CLSM images showed that nanoparticles were delivered into the microconduits created by microneedles and permeated into the epidermis and the dermis. The quantitative determination showed that (i) the permeation of nanoparticles into the skin was enhanced by microneedles, but no nanoparticle reached the receptor solution; (ii) much more nanoparticles deposited in the epidermis than those in the dermis; (iii) the permeation was in a particle size-dependent manner; and (iv) the permeation increased with the nanoparticle concentration increasing until a limit value was reached. These results suggested that microneedles could enhance the intradermal delivery of PLGA nanoparticles. The biodegradable nanoparticles would sustain drug release in the skin and supply the skin with drug over a prolonged period. This strategy would prove to be useful for topical drug administration.

A thermo-sensitive PLGA-PEG-PLGA hydrogel for sustained release of docetaxel

The aim of this study was to investigate the suitability of poly-(d,l-lactic acid-co-glycolic acid) (PLGA)-polyethylene glycol (PEG)-PLGA triblock copolymer as a matrix material for a sustained-release system for docetaxel (DTX). The copolymers were synthesized by ring-opening polymerization reaction and characterized by 1H-NMR and gel permeation chromatography. The DTX-loaded formulations were prepared, characterized. And the antitumor efficacy and the pharmacokinetics of DTX-loaded copolymer on A-549 lung tumor-bearing BALB/cA mice were investigated. The results showed that DTX-loaded copolymer highly increased the solubility of DTX by more than 3000-fold. And copolymer concentration as well as drug loading level exerted appreciable influence on the drug release behavior. Further, the pharmacokinetic test showed that DTX-loaded copolymer could be with the sustained-release nature for over 3 weeks, which correlated well with the in vitro release. Additionally, one intratumoral injection of the thermo-sensitive hydrogel containing DTX was comparable to three intravenous injections of DTX injection in inhibiting the tumor growth in A-549 lung tumor-bearing BALB/cA mice with a less toxic manner. PLGA-PEG-PLGA could thus provide a promising alternate locally delivered vehicle for DTX to achieve prolonged exposure having greater efficacy in inhibiting tumor growth with lower toxicity.

Reducible self-assembling cationic polypeptide-based micelles mediate co-delivery of doxorubicin and microRNA-34a for androgen-independent prostate cancer therapy.

The co-delivery of chemotherapeutic drugs and microRNAs (miR) represents a promising strategy for tumor therapy due to the synergistic effect achieved. In the present study, hydrophobic doxorubicin (DOX) and negatively charged miR-34a were simultaneously delivered via a reducible self-assembling disulfide cross-linked stearyl-peptide-based micellar system (SHRss) using poly(l-arginine)-poly(l-histidine)-stearoyl as the copolymer building unit. The nanoscale SHRss micelles exhibited a low critical micelle concentration (CMC) with positive surface charge. In addition, the present micellar system facilitated the escape of miR-34a from the endosome and release of DOX into the cell nucleus, leading to the downregulation of silent information regulator 1 (SIRT1) expression and inhibition of DU145 and PC3 androgen-independent prostate cancer cell proliferation. In addition, DOX and miR-34a, delivered by SHRss micelles, passively targeted tumor tissue. Furthermore, a synergistic anti-proliferative effect was observed compared with DOX or miR-34a treatment alone in vivo. Our results demonstrate that the SHRss micelles developed in the present study represent a promising approach for combined delivery of gene agents and hydrophobic chemotherapeutic drugs in cancer therapy.

Degradable gene delivery systems based on Pluronics-modified low-molecular-weight polyethylenimine: preparation, characterization, intracellular trafficking, and cellular distribution

Background Cationic copolymers consisting of polycations linked to nonionic amphiphilic block polymers have been evaluated as nonviral gene delivery systems, and a large number of different polymers and copolymers of linear, branched, and dendrimeric architectures have been tested in terms of their suitability and efficacy for in vitro and in vivo transfection. However, the discovery of new potent materials still largely relies on empiric approaches rather than a rational design. The authors investigated the relationship between the polymers’ structures and their biological performance, including DNA compaction, toxicity, transfection efficiency, and the effect of cellular uptake. Methods This article reports the synthesis and characterization of a series of cationic copolymers obtained by grafting polyethyleneimine with nonionic amphiphilic surfactant polyether-Pluronic® consisting of hydrophilic ethylene oxide and hydrophobic propylene oxide blocks. Transgene expression, cytotoxicity, localization of plasmids, and cellular uptake of these copolymers were evaluated following in vitro transfection of HeLa cell lines with various individual components of the copolymers. Results Pluronics can exhibit biological activity including effects on enhancing DNA cellular uptake, nuclear translocation, and gene expression. The Pluronics with a higher hydrophilic-lipophilic balance value lead to homogeneous distribution in the cytoplasm; those with a lower hydrophilic-lipophilic balance value prefer to localize in the nucleus. Conclusion This Pluronic-polyethyleneimine system may be worth exploring as components in the cationic copolymers as the DNA or small interfering RNA/microRNA delivery system in the near future.

Anti-DR5 monoclonal antibody-mediated DTIC-loaded nanoparticles combining chemotherapy and immunotherapy for malignant melanoma: target formulation development and in vitro anticancer activity

Background The increased incidence of malignant melanoma in recent decades, along with its high mortality rate and pronounced resistance to therapy pose an enormous challenge. Novel therapeutic strategies, such as immunotherapy and targeted therapy, are urgently needed for melanoma. In this study, a new active targeting drug delivery system was constructed to combine chemotherapy and active specific immunotherapy. Methods The chemotherapeutic drug, dacarbazine (DTIC), that induces apoptosis through the intrinsic pathway which typically responds to severe DNA damage, was used as a model drug to prepare DTIC-loaded polylactic acid (PLA) nanoparticles (DTIC-NPs), which were covalently conjugated to a highly specific targeting functional TRAIL-receptor 2 (DR5) monoclonal antibody (mAb) that can contribute directly to cancer cell apoptosis or growth inhibition through the extrinsic pathway. Results Our in vitro experiments demonstrated that DTIC-PLA-DR5 mAb nanoparticles (DTIC-NPs-DR5 mAb) are an active targeting drug delivery system which can specifically target DR5-overexpressing malignant melanoma cells and become efficiently internalized. Most strikingly, compared with conventional DTIC-NPs, DTIC-NPs-DR5 mAb showed significantly enhanced cytotoxicity and increased cell apoptosis in DR5-positive malignant melanoma cells. Conclusion The DTIC-NPs-DR5 mAb described in this paper might be a potential formulation for targeting chemotherapy and immunotherapy to DR5-overexpressing metastatic melanoma.

Biodegradable stearylated peptide with internal disulfide bonds for efficient delivery of siRNA in vitro and in vivo.

RNA-based delivery system for cancer therapy remains a challenge. In this study, a stearyl-peptide (SHR) was synthesized using arginine, histidine, cysteine, and stearyl moieties. Further, the stearyl-peptides were cross-linked by disulfide bonds to obtain cross-linked polypeptides (SHRss) with different molecular weight (SHRss1, SHRss2, SHRss3, SHRss4). The SHRss could effectively condense small interfering RNA (siRNA) into polyplexes with a hydrodynamic size of 100-300 nm and zeta potential of 20-40 mV. Flow cytometry and confocal laser scanning microscope studies revealed high cellular uptake and rapid dissociation behavior of SHRss2/siRNA complexes. Long-lasting high concentration of siRNA in cytoplasm was observed even at 24 h after SHRss2/Cy3-siRNA transfection. Compared with SHR, the SHRss showed much improved siRNA interference efficiency targeting luciferase on Luc-Hela cells. Moreover, SHRss2 exhibited higher interference efficiency and slower decay rate on Luc-Hela cells than Lipofectamine 2000 and SHR. In addition, much weaker expression of red fluorescence protein was also observed on SHRss2/simCh-treated mCherry-HEK293 cells than Lipofectamine 2000 and SHR. The SHRss did not induce cytotoxicity at siRNA concentrations of 25-200 nM under transfection. The in vivo studies demonstrated the gene interference efficiency of SHRss2/siRNA complexes. Our studies indicated that the SHRss are promising and efficient nonviral vectors for siRNA delivery.

Ocular delivery of cyclosporine A based on glyceryl monooleate/poloxamer 407 liquid crystalline nanoparticles: preparation, characterization, in vitro corneal penetration and ocular irritation

The purpose of this study was to develop an ophthalmic drug delivery system for cyclosporine A (CsA) based on glyceryl monooleate (GMO)/poloxamer 407 liquid crystalline nanoparticles with reduced ocular irritancy and improved corneal penetration. CsA-loaded liquid crystalline nanoparticles were prepared via fragmentation of a bulk GMO/poloxamer 407 cubic phase gel by high-pressure homogenization and characterized. Corneal permeation and retention was evaluated using modified Franz diffusing cells. Intra-corneal transportation was investigated with fluorescein isothiocyanate (FITC)-labeled liquid crystalline nanoparticles. Ocular irritation was then evaluated using the Draize method. The mean particle size of liquid crystalline nanoparticles was 193.5 nm and the entrapment efficiency was 95.11 ± 0.67%. A bicontinuous cubic phase of cubic P-type was determined using cryo-transmission electron microscopy (cryo-TEM) observation and small angle X-ray diffraction (SAXD) analysis. A 1.52-fold increase in Js and a 2.2-fold increase in corneal retention was achieved by liquid crystalline nanoparticles compared with oil solution. In vitro corneal permeation investigated with FITC-labeled liquid crystalline nanoparticles revealed that CsA penetrated across the cornea under the transportation of liquid crystalline nanoparticles. Liquid crystalline nanoparticles exhibited excellent ocular tolerance in the ocular irritation test. This low-irritant vehicle based on liquid crystalline nanoparticles might be a promising system for effective ocular CsA delivery.

Degradable polyethylenimine derivate coupled to a bifunctional peptide R13 as a new gene-delivery vector

Background To solve the efficiency versus cytotoxicity and tumor-targeting problems of polyethylenimine (PEI) used as a nonviral gene delivery vector, a degradable PEI derivate coupled to a bifunctional peptide R13 was developed. Methods First, we synthesized a degradable PEI derivate by crosslinking low-molecular-weight PEI with pluronic P123, then used tumor-targeting peptide arginine-glycine-aspartate-cysteine (RGDC), in conjunction with the cell-penetrating peptide Tat (49–57), to yield a bifunctional peptide RGDC-Tat (49–57) named R13, which can improve cell selection and increase cellular uptake, and, lastly, adopted R13 to modify the PEI derivates so as to prepare a new polymeric gene vector (P123-PEI-R13). The new gene vector was characterized in terms of its chemical structure and biophysical parameters. We also investigated the specificity, cytotoxicity, and gene transfection efficiency of this vector in αvβ3-positive human cervical carcinoma Hela cells and murine melanoma B16 cells in vitro. Results The vector showed controlled degradation, strong targeting specificity to αvβ3 receptor, and noncytotoxicity in Hela cells and B16 cells at higher doses, in contrast to PEI 25 KDa. The particle size of P123-PEI-R13/DNA complexes was around 100–250 nm, with proper zeta potential. The nanoparticles can protect plasmid DNA from being digested by DNase I at a concentration of 6 U DNase I/μg DNA. The nanoparticles were resistant to dissociation induced by 50% fetal bovine serum and 600 μg/mL sodium heparin. P123-PEI-R13 also revealed higher transfection efficiency in two cell lines as compared with PEI 25 KDa. Conclusion P123-PEI-R13 is a potential candidate as a safe and efficient gene-delivery carrier for gene therapy.

Study on the prostate cancer-targeting mechanism of aptamer-modified nanoparticles and their potential anticancer effect in vivo

Ligand-mediated prostate cancer (PCa)-targeting gene delivery is one of the focuses of research in recent years. Our previous study reported the successful preparation of aptamer-modified nanoparticles (APT-NPs) in our laboratory and demonstrated their PCa-targeting ability in vitro. However, the mechanism underlying this PCa-targeting effect and their anticancer ability in vivo have not yet been elucidated. The objective of this study was to assess the feasibility of using APT-NPs to deliver micro RNA (miRNA) systemically to PCa cells, to testify their tumor-targeting efficiency, and to observe their biodistribution after systemic administration to a xenograft mouse model of PCa. In addition, the effect of APT depletion and endocytosis inhibitors on cellular uptake was also evaluated quantitatively in LNCaP cells to explore the internalization mechanism of APT-NPs. Finally, blood chemistry, and renal and liver function parameters were measured in the xenograft mouse model of PCa to see whether APT-NPs had any demonstrable toxicity in mice in vivo. The results showed that APT-NPs prolonged the survival duration of the PCa tumor-bearing mice as compared with the unmodified NPs. In addition, they had a potential PCa-targeting effect in vivo. In conclusion, this research provides a prototype for the safe and efficient delivery of miRNA expression vectors to PCa cells, which may prove useful for preclinical and clinical studies on the treatment of PCa.