Self-assembling HA/PEI/dsRNA-p21 ternary complexes for CD44 mediated small active RNA delivery to colorectal cancer

Our previous work proved that sequence specific double strand RNA (dsRNA-p21) effectively activated p21 gene expression of colorectal cancer (CRC) cells and consequently suppressed CRC growth. However, efficient delivery system is a significant challenge to achieve sufficient therapy. In this study, a self-assembled HA/PEI/dsRNA-p21 ternary complex (TC-dsRNA-p21) was developed for the tumor-target delivery of dsRNA-p21 into CRC cells. Hyaluronic acid (HA) was introduced to shield the PEI/dsRNA-p21 binary complexes (BC-dsRNA-p21) for reducing the cytotoxicity of PEI and for increasing the tumor-targeted intracellular uptake by cancer cells through HA-CD44 mediated endocytosis. Comparing to the BC-dsRNA-p21, the TC-dsRNA-p21 showed increase in size, decrease in zeta potential, low cytotoxicity as well as high stability in physiological conditions due to the anionic shielding. Confocal microscopy analysis and flow cytometry confirmed that TC-dsRNA-p21 had high transfection efficiency in the CD44-abundant Lovo cells, as compared with binary complex. In vitro physiological experiment showed that, comparing to the control group, the TC-dsRNA-p21 effectively activated the expression of p21 mRNA and P21 protein, causing blockage of cell cycle at G₀/G₁ phase and suppression of cancer cell proliferation as well as colony formation. Furthermore, in vivo distribution experiment demonstrated that the TC-dsRNA-p21 could effectively accumulate at rectal wall for up to 10?h, following in situ application. These findings indicated that TC-dsRNA-p21 might hold great potential for delivering dsRNA-p21 to treat CRC.     

藻酸盐/PEI/DNA复合载体作为一种新型基因递送系统

目的克服多聚乙烯亚胺(PEI，polyethlenimine)/DNA载体对细胞的毒性以及在含血清培养基里对癌细胞基因的转移率低的问题。方法利用具有水溶性、可生物降解的、并带有负电的藻酸盐(alginate)对PEI/DNA载体进行包衣，制备出复合载体，并在体外含50%血清培养基里，与PEI/DNA载体比较对C3癌细胞转染率。结果 在含50%血清的培养基里，藻酸盐包衣制备的复合体载体［alginate:DNA，0.15 (w/w);PEI:DNA，N:P=10］与PEI/DNA载体相比，对C3癌细胞基因转染率高出10～30倍，而且其表面正电荷数比PEI/DNA载体减少了一半，颗粒较小，并降低对细胞毒性和红血球集聚反应。结论作为新型的藻酸盐包衣制备的复合载体能提高在体外含高浓度血清培养基里对C3癌细胞的转染率，并能减少其对细胞毒性。     

Efficient in vivo gene transfection by stable DNA/PEI complexes coated by hyaluronic acid

Plasmid DNA was mixed with polyethyleneimine (PEI) and hyaluronic acid (HA) to afford ternary complexes with negative surface charge regardless of the mixing order. They showed reduced non-specific interactions with blood components. When DNA and PEI were mixed at a high concentration such as that used in in vivo experiments, they soon aggregated, and large particles were formed. On the other hand, pre-addition of HA to DNA prior to PEI effectively diminished the aggregation, and 10% (in volume) of the complexes remained as small particles with a diameter below 80 nm. Those negatively charged small ternary complexes induced a much stronger extra-gene expression in tumor than binary DNA/PEI complex after intratumoral or intravenous injection into the mice bearing B16 cells.     

Nanostructured hyaluronic acid-based materials for active delivery to cancer

Importance of the field: Active targeting of bioactive molecules by physicochemical association with hyaluronic acid (HA) is an attractive approach in current nanomedicine because HA is biocompatible, non-toxic and non-inflammatory.

Areas covered in this review: This review focuses on synthesis, physicochemical characterization and biological properties of different nanoparticulate delivery systems that include HA in their structures. Chemically based approaches to the delivery of small molecule drugs, proteins and nucleic acids in which they become chemically or physically bound to hyaluronic acid are reviewed, including the use of molecular HA conjugates and nanocarriers. The systems are considered in terms of intracellular delivery to different cultured cells that express HA-specific receptors (hyaladherines) differently. The in vivo biodistribution and therapeutic effect of these systems are discussed.

What the reader will gain: Different synthetic methodologies for preparations of HA-based nanoparticles are presented extensively. HA nanoparticulate systems of various structures can be compared with respect to their in vitro assays and in vivo biodistribution.

Take home message: To make HA useful as an intravenous targeting carrier, strategies have to be devised to: reduce HA clearance from the blood; suppress the HA uptake by liver and spleen; and provide tumor-triggered mechanisms of release of an active drug from the HA carrier.     

DNA/RNA-based formulations for treatment of breast cancer

Introduction: To develop a successful formulation for the gene therapy of breast cancer, an effective therapeutic nucleic acid and a proper delivery system are essential. Increased understanding of breast cancer, and developments in biotechnology, material science and nanotechnology have provided a major impetus in the development of effective formulations for the gene therapy of breast cancer.

Areas covered: We discuss DNA/RNA-based formulations that can inhibit the growth of breast cancer cells and control the progress of breast cancer. Targets for the gene therapy of breast cancer, DNA/RNA-based therapeutics and delivery systems are summarized. And examples of successful DNA/RNA-based formulations for breast cancer gene therapy are reviewed.

Expert opinion: Several challenges remain in developing effective DNA/RNA-based formulations for treatment of breast cancer. Firstly, most of the currently utilized targets are not effective enough as monotherapy for breast cancer. Secondly, the requirements for co-delivery system make the preparation of formulation more complicated. Thirdly, nanoparticles with the modification of tumor-targeting ligands could be more unstable in circulation and normal tissues. Lastly, immune responses against the viral vectors are unfavorable for the gene therapy of breast cancer because of the damage to the host and the impaired therapeutic ability.     

Ovarian cancer targeted hyaluronic acid-based nanoparticle system for paclitaxel delivery to overcome drug resistance

Abstract

Objective: Most primary human ovarian tumors and peritoneal implants, as well as tumor vascular endothelial cells, express the CD44 family of cell surface proteoglycans, the natural ligand for which is hyaluronic acid (HA). Paclitaxel (PTX) is an effective chemotherapeutic agent that is widely used for the treatment of several cancers, including ovarian cancer. This study aimed to develop a HA-based PTX-loaded nanoparticle system to improve the ovarian cancer therapeutic effects.

Methods: PTX-loaded cationic nanostructured lipid nanoparticles (PTX-NLCs) were prepared. HA-PE was then coated onto the PTX-NLCs by electrostatic adsorption to form HA-PTX-NLCs. In vitro tumor cell inhibition efficiency was analyzed on SKOV3 human ovarian cancer cells (SKOV3 cells) and PTX-resistant SKOV3 cells (SKOV3/PTX cells). In vivo anticancer ability was evaluated with mice bearing SKOV3 ovarian cancer cells xenografts.

Results: HA-PTX-NLCs had an average diameter of 163?nm, and PTX was incorporated with an efficiency of over 80%. The in vitro viability of SKOV3 cells and SKOV3/PTX cells was obviously inhibited by HA-PTX-NLCs. In the ovarian cancer cells model, significant reduction in tumor growth was observed, whereas the conventional PTX injection group did not achieve significance.

Conclusion: This study demonstrated that significantly improved results were obtained by the newly constructed HA-PTX-NLCs, in terms of in vitro and in vivo therapeutic efficacy. These findings strongly support the superiority of HA based nano-system for the PTX delivery, thus enhance the efficacy of ovarian cancer chemotherapy.     

Polymer-based non-viral gene delivery as a concept for the treatment of cancer

Gene therapy has become a promising technique for the treatment of cancer. Nevertheless, the success of gene therapy depends on the effectiveness of the vector. The challenge of a gene carrier is to deliver exogenous DNA from the site of administration into the nucleus of the appropriate target cell. Polymer-based vectors are biologically safe, have low production costs and are efficient tools for gene therapy. Although non-degradable polyplexes exhibit high gene expression levels, their application potential is limited due to their inability to be effectively eliminated, which results in cytotoxicity. The development of biodegradable polymers has allowed for high levels of transfection without cytotoxicity. For site-specific targeting of polyplexes, further modifications, such as incorporation of ligands, can be performed. Most expectations have been addressed to polyplexes architecture according it dynamic response with the microenvironment.     

An update on calcium carbonate nanoparticles as cancer drug/gene delivery system

Introduction: In recent years, the applications of calcium carbonate (CaCO₃) nanoparticles (NPs) have gained extensive interest as targeted drug/gene delivery systems to cancerous tissues and cells due to their accessibility, low cost, safety, biocompatibility, pH-sensitivity, and slow biodegradability.

Areas covered: Drug-loaded CaCO₃ NPs (CCNPs) have been reviewed. An updated search on the current state of CCNPs as cancer drug/gene delivery systems with a focus on their special properties including pH-sensitivity, biodegradability, and sustained release performance has been also assessed.

Expert opinion: Based on the reviewed literature, CCNPs, because of their superior features, will have a great aiding role in safe and efficient cancer treatment in the near future.     

Poly(amidoamine) dendrimer complexes as a platform for gene delivery

Introduction: Gene therapy is one of the most effective ways to treat major infectious diseases, cancer and genetic disorders. It is based on several viral and non-viral systems for nucleic acid delivery. The number of clinical trials based on application of non-viral drug and gene delivery systems is rapidly increasing.

Areas covered: This review discusses and summarizes recent advances in poly(amidoamine) dendrimers as effective gene carriers in vitro and in vivo, and their advantages and disadvantages relative to viral vectors and other non-viral systems (liposomes, linear polymers) are considered.

Expert opinion: In this regard, dendrimers are non-immunogenic and have the highest efficiency of transfection among other non-viral systems, and none of the drawbacks characteristic for viral systems. The toxicity of dendrimers both in vitro and in vivo is an important question that has been addressed on many occasions. Several non-toxic and efficient multifunctional dendrimer-based conjugates for gene delivery, along with modifications to improve transfection efficiency while decreasing cytotoxicity, are discussed. Twelve paradigms that affected the development of dendrimer-based gene delivery are described. The conclusion is that dendrimers are promising candidates for gene delivery, but this is just the beginning and further studies are required before using them in human gene therapy.     

多柔比星纳米递药系统在三阴性乳腺癌靶向治疗中的应用

三阴性乳腺癌为高度恶性肿瘤。多柔比星是三阴性乳腺癌的常规化疗药物,该药药理作用是通过嵌入DNA碱基对之间,干扰基因转录,抑制mRNA和DNA合成。常规给药方式对正常组织损伤严重。多柔比星纳米递药系统借助肿瘤酸性微环境实现缓控释效应,多柔比星与肿瘤细胞的组织相容性增加,对正常组织影响较小。该系统有效抑制和杀灭肿瘤细胞,明显减轻正常细胞的细胞毒性。本文综述了近年来多柔比星靶向纳米递药系统的应用,以期开拓三阴性乳腺癌的的靶向治疗新视野。     

Targeted gene delivery to hepatoma cells using galactosylated liposome-polycation-DNA complexes (LPD)

A major goal for gene therapy is to obtain targeted vectors that transfer genes efficiently to specific cell types. The liver possesses a variety of characteristics that make this organ very attractive for gene therapy. In the present study, four cholesterylated thiogalactosides 1a ～ d with different spacer length were synthesized to formulate novel lipid-polycation-DNA (LPD) complexes, which were composed of galactosylated cationic liposomes, protamine sulfate and plasmid DNA. The galactosylated LPD1c significantly improved the levels of gene expression in cultured hepatoma cells HepG2 and SMMC-7721, while LPD1a and LPD1b did not significantly improve the levels compared with non-galactosylated LPD. Meanwhile, increased transfection activity was not observed in mouse fibroblasts L929 for galactosylated LPDs. Cytotoxicity of galactosylated LPDs assay showed they had no obvious toxicities to L929 cells and HepG2 cells. In summary, the length of the spacer between the anchor and galactose residues was important for the recognition of asialoglycoprotein receptor. The LPD1c described here, combining the condensing effect of protamine and the targeting capability of cholesterylated thiogalactosides, are potentially useful gene carriers to liver parenchymal cells.     

Multifunctional nanoemulsions for intraductal delivery as a new platform for local treatment of breast cancer

Considering that breast cancer usually begins in the lining of the ducts, local drug administration into the ducts could target cancers and pre-tumor lesions locally while reducing systemic adverse effects. In this study, a cationic bioadhesive nanoemulsion was developed for intraductal administration of C6 ceramide, a sphingolipid that mediates apoptotic and non-apoptotic cell death. Bioadhesive properties were obtained by surface modification with chitosan. The optimized nanoemulsion displayed size of 46.3?nm and positive charge, properties that were not affected by ceramide encapsulation (0.4%, w/w). C6 ceramide concentration necessary to reduce MCF-7 cells viability to 50% (EC₅₀) decreased by 4.5-fold with its nanoencapsulation compared to its solution; a further decrease (2.6-fold) was observed when tributyrin (a pro-drug of butyric acid) was part of the oil phase of the nanocarrier, a phenomenon attributed to synergism. The unloaded nanocarrier was considered safe, as indicated by a score <0.1 in HET-CAM models, by the high survival rates of Galleria mellonella larvae exposed to concentrations ≤500?mg/mL, and absence of histological changes when intraductally administered in rats. Intraductal administration of the nanoemulsion prolonged drug localization for more than 120?h in the mammary tissue compared to its solution. These results support the advantage of the optimized nanoemulsion to enable mammary tissue localization of C6 ceramide.     

P53-Derived peptides conjugation to PEI: an approach to producing versatile and highly efficient targeted gene delivery carriers into cancer cells

ABSTRACT

Objectives: Targeted delivery of cytotoxic drugs or therapeutic antisense RNAs into specific cells is a major bottleneck in cancer therapy. To overcome this problem and improve the specificity for cancer cells, we describe a new-targeted delivery system using p53-derived peptides, namely PNC 27 and PNC 28. These peptides target HDM-2 on the surface of cancer cells. HDM-2 is overexpressed on the surface of cancerous cells, but not present on the untransformed cells.

Methods: To determine HDM-2-expressing cells, we used immunocytochemistry and flow cytometry analysis on nine cell lines including MCF-7 and NIH-3t3. Conjugation of peptides to vectors was confirmed using reverse-phase high-pressure liquid chromatography (RP-HPLC). Physicochemical properties of vector/DNA complexes including particle size, surface charge and DNA condensation ability were determined. In transfection studies, three plasmids were used including luciferase, pEGFP and shRNA plasmid against Bcl-XL mRNA. The level of Bcl-XL expression was determined by real-time PCR and western blot techniques.

Results: The results of gene delivery and shRNA-based gene silencing studies indicated that conjugation of PNC peptides could enhance gene delivery efficiently with high-targeted activity exclusively into cancer cells.

Conclusion: Our results strongly indicated that this targeting system could be utilized as an efficient targeting method for most cancer cells.     

Mitoxantrone as a first-line treatment of advanced breast cancer

Summary Forty-two women with measurable or evaluable advanced breast cancer who had received neither prior chemotherapy for advanced disease nor any anthracycline-containing regimen as adjuvant were entered in a phase II study of mitoxantrone (Novantrone^®; dihydroxyanthracenedione). Patients were aged from 36 to 80 years, performance status was from 0 to 2. All patients had normal hematological status and normal renal and liver function tests. Cardiac scintigraphy and sonography techniques were used to monitor cardiac function. Mitoxantrone was administered at a dose of 14 mg/m² in 100 ml 5% dextrose solution over 30 minutes, repeated every three weeks.The number of courses per patient ranged from 2 to 12. Of 42 eligible patients 39 were fully evaluable for response and all for drug toxicity. Responses to treatment were: complete response four patients, partial response 10 patients, stable disease 18 patients and progressive disease seven patients. The overall response rate was 36% (95% confidence limits 20–52%). Three patients showed decreased left ventricular ejection fraction but no patient developed signs of overt left ventricular failure during the treatment period. Hematological and gastrointestinal toxicities were mild. Hair loss was minimal.The data indicate that mitoxantrone is an effective agent for the treatment of advanced breast cancer with mild side-effects, especially with respect to nausea/vomiting, hair loss and cardiotoxicity.     

Controlled release of PEI/DNA complexes from PLGA microspheres as a potent delivery system to enhance immune response to HIV vaccine DNA prime/MVA boost regime.

A novel approach involving the preparation of biodegradable PLGA microspheres containing entrapping complexes of DNA and polyethylenimine was developed to improve the delivery of DNA into antigen-presenting cells after intramuscular injection. Compared to the traditional biodegradable microspheres which release naked DNA, these microspheres released intact and penetrative PEI/DNA complexes over a period of 2 weeks in vitro. In addition, the DNA was not degraded during encapsulation in the PLGA microspheres, owing to the protection of polyethylenimine. After i.m. immunization, the microspheres induced significantly enhanced serum antibody responses 2-3 orders of magnitude greater than naked DNA. Additionally, in contrast to naked DNA, the microspheres induced potent CTL responses at a low dose.     

Dextran-coated iron oxide nanoparticle for delivery of miR-29a to breast cancer cell line

In the last years, miRNAs have been associated with molecular pathways of cancer and other diseases. The change of expression level of miRNA has an inhibitory role in tumorigenesis. Nevertheless, the poor bioavailability of miRNA due to the rapid enzymatic degradation is a critical handicap in cancer therapy. In this study, we designed dextran-coated iron oxide-based nanoparticle for the delivery of miR-29a to breast cancer cells and analyzed its therapeutic efficacy in vitro. Results indicated that the presence of dextran-coated magnetic nanoparticles, loaded with miR29a, enhanced the selective delivery of miR-29a. Further, miR-29a complex nanoparticles caused down-regulation of anti-apoptotic genes. These results pave the way for further investigations into the possible use of miR-29a complex magnetic nanoparticles for breast cancer therapy.     

The Hedgehog signalling pathway as a therapeutic target in early breast cancer development

The Hedgehog (Hh) signalling pathway is a highly conserved developmental pathway, which plays critical roles in patterning of the embryo through epithelial to mesenchymal signalling and the maintenance of stem cells in the adult organism. There is increasing evidence that this pathway is dysregulated in many malignancies, including breast cancer. While there has been a significant decrease in mortality from breast cancer, a number of treatment challenges remain, particularly in those tumours which develop resistance to endocrine-based therapy, or which lack expression of hormone or c-erbB2/HER2 receptors. Therapeutic manipulation of the Hh pathway as a potential cancer therapy is attracting great interest, with preclinical studies and clinical trials underway in a range of malignancies. This review highlights important recent developments that affect the potential of the Hh pathway as a novel therapeutic target in early breast cancer.     

Therapeutic efficacy of folate receptor-targeted amphiphilic cyclodextrin nanoparticles as a novel vehicle for paclitaxel delivery in breast cancer

Purpose: The aim of this study is to test folate-conjugated cyclodextrin nanoparticles (FCD-1 and FCD-2) as a vehicle for reducing toxicity and increasing the antitumor efficacy of paclitaxel especially for metastatic breast cancer.

Methods: For the evaluation of PCX-loaded FCD nanoparticles, animal studies were realised in terms of survival rate, tumour size, weight change, metastazis and histopathological examination.

Results: FCD-1 displayed significant advantages such as efficient targeting of folate receptor positive breast cancer cells and having considerably lower toxicity compared to that of Cremophor^®. When loaded with paclitaxel, FCD-1 nanoparticles, which have smaller particle size, neutral zeta potential, high encapsulation efficiency and better loading capacity for controlled release, emerged as an effective formulation in terms of cytotoxicity and high cellular uptake. In an experimental breast cancer model, anticancer activity of these nanoparticles were compatible with that of paclitaxel in Cremophor^® however repeated administrations of FCD-1 nanoparticles were better tolerated by the animals. These nanoparticles were able to localise in tumour site. Both paclitaxel-loaded FCD-1 and FCD-2 significantly reduced tumour burden while FCD-1 significantly improved the survival.

Conclusions: Folate-conjugated amphiphilic cyclodextrin nanoparticles can be considered as promising Cremophor^®-free, low-toxicity and efficient active drug delivery systems for paclitaxel.     

适配体C2min介导的可靶向2种前列腺癌基因的递送系统

目的 通过合成可靶向两种前列腺癌的基因载体PAMAM-PEG-C2min,以提高基因的转染效率和肿瘤靶向性。方法 将双功能聚乙二醇的一端与聚酰胺-胺（PAMAM）相连,另一端与适配体（C2min）连接,并利用¹H NMR技术对合成的PAMAM-PEG-C2min基因载体进行结构鉴定。通过两种前列腺癌PC3和LNCaP细胞的体外摄取和基因转染实验（包载siR-M基因）,考察纳米复合物的生物学特性。并利用动物活体成像技术考察合成的纳米复合物的体内分布特征。结果 核磁共振结果表明,本研究成功合成了PAMAM-PEG-C2min。PC3和LNCaP细胞对PAMAM-PEG-C2min的摄取结果体现出浓度依赖性。且与不经C2min修饰的PAMA-PEGM相比,PAMAM-PEG-C2min递药系统的基因转染效率和肿瘤细胞靶向性明显提高。体内靶向性结果表明,PAMAM-PEG-C2min可实现同时靶向2种前列腺癌组织的作用。结论 本研究合成的PAMAM-PEG-C2min递送载体具有良好的肿瘤靶向性,为前列腺癌的综合治疗和靶向治疗提供了新的技术平台。