Search

Hao Wu,1,2 Yanhong Liu,2 Liqing Chen,2 Shuangqing Wang,1,2 Chao Liu,2 Heming Zhao,2 Mingji Jin,1,2 Shuangyan Chang,2 Xiuquan Quan,3 Minhu Cui,3 Hongshuang Wan,2,3 Zhonggao Gao,1,2 Wei Huang2 1Key Laboratory of Natural Medicines of the Changbai Mountain, Ministry of Education, College of Pharmacy, Yanbian University, Yanji, Jilin Province, 133002, People’s Republic of China; 2State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Department of Pharmaceutics, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China; 3Department of Gastroenterology, Yanbian University Hospital, Yanji, Jilin Province, 133000, People’s Republic of ChinaCorrespondence: Zhonggao Gao; Wei Huang, Tel +86 10 63028096 ; +86 10 63026505, Email zggao@imm.ac.cn; huangwei@imm.ac.cnIntroduction: The blood–brain barrier (BBB) is a key obstacle to the delivery of drugs into the brain. Therefore, it is essential to develop an advanced drug delivery nanoplatform to solve this problem. We previously screened a small rabies virus glycoprotein 15 (RVG15) peptide with 15 amino acids and observed that most of the RVG15-modified nanoparticles entered the brain within 1 h of administration. The high BBB penetrability gives RVG15 great potential for brain-targeted drug delivery systems. Moreover, a multifunctional integrated nanoplatform with a high drug-loading capacity, tunable functionality, and controlled drug release is crucial for tumor treatment. Zeolitic imidazolate framework (ZIF-8) is a promising nanodrug delivery system.Methods: Inspired by the biomimetic concept, we designed RVG15-coated biomimetic ZIF-8 nanoparticles (RVG15-PEG@DTX@ZIF-8) for docetaxel (DTX) delivery to achieve efficient glioblastoma elimination in mice. This bionic nanotherapeutic system was prepared by one-pot encapsulation, followed by coating with RVG15-PEG conjugates. The size, morphology, stability, drug-loading capacity, and release of RVG15-PEG@DTX@ZIF-8 were thoroughly investigated. Additionally, we performed in vitro evaluation, cell uptake capacity, BBB penetration, and anti-migratory ability. We also conducted an in vivo evaluation of the biodistribution and anti-glioma efficacy of this bionic nanotherapeutic system in a mouse mode.Results: In vitro studies showed that, this bionic nanotherapeutic system exhibited excellent targeting efficiency and safety in HBMECs and C6 cells and high efficiency in crossing the BBB. Furthermore, the nanoparticles cause rapid DTX accumulation in the brain, allowing deeper penetration into glioma tumors. In vivo antitumor assay results indicated that RVG15-PEG@DTX@ZIF-8 significantly inhibited glioma growth and metastasis, thereby improving the survival of tumor-bearing mice.Conclusion: Our study demonstrates that our bionic nanotherapeutic system using RVG15 peptides is a promising and powerful tool for crossing the BBB and treating glioblastoma.Keywords: glioblastoma, blood–brain barrier, RVG15, biomimetic nanoparticle, zeolitic imidazolate frameworks-8, ZIF-8

Background Glioma is the most common primary malignant brain tumor with a dreadful overall survival and high mortality. One of the most difficult challenges in clinical treatment is that most drugs hardly pass through the blood–brain barrier (BBB) and achieve efficient accumulation at tumor sites. Thus, to circumvent this hurdle, developing an effectively traversing BBB drug delivery nanovehicle is of significant clinical importance. Rabies virus glycoprotein (RVG) is a derivative peptide that can specifically bind to nicotinic acetylcholine receptor (nAChR) widely overexpressed on BBB and glioma cells for the invasion of rabies virus into the brain. Inspired by this, RVG has been demonstrated to potentiate drugs across the BBB, promote the permeability, and further enhance drug tumor-specific selectivity and penetration. Methods Here, we used the RVG15, rescreened from the well-known RVG29, to develop a brain-targeted liposome (RVG15-Lipo) for enhanced BBB permeability and tumor-specific delivery of paclitaxel (PTX). The paclitaxel-cholesterol complex (PTX-CHO) was prepared and then actively loaded into liposomes to acquire high entrapment efficiency (EE) and fine stability. Meanwhile, physicochemical properties, in vitro and in vivo delivery efficiency and therapeutic effect were investigated thoroughly. Results The particle size and zeta potential of PTX-CHO-RVG15-Lipo were 128.15 ± 1.63 nm and −15.55 ± 0.78 mV, respectively. Compared with free PTX, PTX-CHO-RVG15-Lipo exhibited excellent targeting efficiency and safety in HBMEC and C6 cells, and better transport efficiency across the BBB in vitro model. Furthermore, PTX-CHO-RVG15-Lipo could noticeably improve the accumulation of PTX in the brain, and then promote the chemotherapeutic drugs penetration in C6luc orthotopic glioma based on in vivo imaging assays. The in vivo antitumor results indicated that PTX-CHO-RVG15-Lipo significantly inhibited glioma growth and metabasis, therefore improved survival rate of tumor-bearing mice with little adverse effect. Conclusion Our study demonstrated that the RVG15 was a promising brain-targeted specific ligands owing to the superior BBB penetration and tumor targeting ability. Based on the outstanding therapeutic effect both in vitro and in vivo, PTX-CHO-RVG15-Lipo was proved to be a potential delivery system for PTX to treat glioma in clinic., Graphical Abstract

Mingji Jin,1,2,* Yan Hou,1– 3,* Xiuquan Quan,1,2,4,* Liqing Chen,1,2 Zhonggao Gao,1,2 Wei Huang1,2 1State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China; 2Beijing Key Laboratory of Drug Delivery Technology and Novel Formulations, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100050, People’s Republic of China; 3Department of Pharmacy, Yanbian University, Yanji, Jilin, 133000, People’s Republic of China; 4Department of Emergency Medicine, Affiliated Hospital of Yanbian University, Yanji, Jilin, 133000, People’s Republic of China*These authors contributed equally to this workCorrespondence: Zhonggao Gao; Wei Huang Tel +86 010 63028096; +86 010 63026505Email zggao@imm.ac.cn; huangwei@imm.ac.cnBackground: The dual-loaded nano-delivery system can realize chemotherapeutic drug and small interfering RNA (siRNA) co-loading as well as enhance the therapeutic effect of drugs on tumors through a synergistic effect, while reducing their toxic and side effects on normal tissues.Methods: Previously, we developed layered smart nanoparticles (NPs) to co-deliver survivin siRNA as well as small molecule drugs for lung cancer. In this study, we used such smart NPs to co-deliver paclitaxel (PTX) and siRNA against vascular endothelial growth factor (VEGF) gene for breast cancer therapy in mice models. For the prepared NPs, characterizations such as particle size, zeta potential, gel electrophoresis imaging and in vitro stability were investigated. Then, 4T1 cells were used to evaluate the in vitro VEGF silencing capacity, tumor cell inhibitory and anti-apoptotic abilities. Finally, an orthotopic model of mouse breast cancer was established to evaluate the in vivo antitumor effects and safety properties of PTX-siRNAVEGF-NPs.Results: We prepared PTX-siRNAVEGF-NPs with particle size of 85.25 nm, PDI of 0.261, and zeta potential of 5.25 mV. The NPs with VEGF siRNA effectively knocked down the expression of VEGF mRNA. Cell counting kit-8 (CCK-8) and apoptosis assays revealed that the PTX-siRNAVEGF-NPs exhibited antiproliferation effect of PTX on 4T1 cells. The in vivo anti-tumor study indicated that PTX-siRNAVEGF-NPs could exert an antitumor effect by inhibiting the formation and development of new blood vessels in tumor tissues, thereby cutting off nutrient and blood supplies required for tumor tissue growth. Both the anti-tumor efficacy and in vivo safety of the PTX-siRNAVEGF-NPs group were better than that of the PTX-NPs and siRNAVEGF-NPs groups.Conclusion: The combination of PTX and VEGF siRNA exerts good antitumor effect on 4T1 tumor cells. This study provides a theoretical and practical basis for breast cancer therapy.Keywords: paclitaxel, VEGF siRNA, breast cancer, co-delivery

Glioblastoma (GBM) is an aggressive malignancy and therapeutic options are limited due to the presence of the blood-brain barrier (BBB). RVG-29, a 29-amino-acid polypeptide derived from the rabies virus glycoprotein (RVG), has excellent brain-targeted capacity across the BBB. We reduced the size of this peptide to get a15-amino-acid polypeptide (RVG-15), while retaining its brain-targeted capacity across the BBB. First, we synthesized a novel nanocarrier RVG-15-PEG2000-DSPE. Next, DOX-loaded polymeric micelles (DOX RVG-15-PMs) were prepared in an electrostatic interaction-dependent manner. Finally, we evaluated its antitumor benefits in vitro at the cellular level and in vivo using an in situ tumour-bearing mouse model. MALDI-TOF-MS and FTIR spectra confirmed the successful synthesis of the novel nanocarrier. The prepared DOX RVG-15-PMs displayed even size distribution, a high entrapment efficiency and satisfactory in vitro release behaviour. In vitro blank RVG-15-PMs were excellent, safe and highly biocompatible as drug delivery carriers. DOX-loaded micelles were easily taken up by C6 cells and could effectively inhibit cancer development and metastasis. In vivo, DOX RVG-15-PMs delayed weight loss, prevented cancer cell metastasis and accelerated cancer cell apoptosis in tumour-bearing mice. Our novel brain-targeted nanocarrier is highly feasible, while DOX RVG-15-PMs exert significant antiglioma effects, both in vitro and in vivo.

Phenylboronic acid (PBA)-mediated tumor targeting nanovector is an attractive strategy for enhancing siRNA delivery and treatment of metastatic cancers. However, its nonspecific binding with various biological membranes containing cis-diol moieties restricts its potential application by systematic administration. Herein, we constructed a novel pH-activated “sheddable” PEG-coated nanoparticle for effective treatment of primary tumors and metastases, which was based on the conjugation of catechol group modified poly(ethylene glycol) (PEG-Cat) and PBA-terminated polyethylenimine (PEI-PBA) via the borate ester formed between PBA and Cat. By virtue of the pH-dependent stability of borate ester in an aqueous medium, the PEG-shell could “shield” the PBA ligand in systemic circulation to reduce its “off-target effect”, while PEG was detached at tumor extracellular pH (~6.5) to expose intact PBA moiety. Simultaneously, the PBA ligand could bind with overexpressed sialic acid residues on cancer cells, giving rise to enhanced cellular internalization. In addition, the PBA moieties could also couple with each 3'-end ribose of double-stranded siRNA. siRNAs were used as both a payload and a pH-responsive intermolecular cross-linker, and thereby acquired sufficient stability during circulating in blood and a rapidly triggered release in response to acidic endosomal/lysosomal pH-stimuli. As a result, this dual pH-sensitive nanoparticle showed enhanced siRNA uptake, gene silencing efficacy and anti-metastatic effects in vitro. Furthermore, in vivo studies demonstrated that PBA-based nanoparticles effectively accumulated in tumor and inhibited tumor growth and metastasis in 4T1 orthotopic mammary tumor model after intravenous administration.

Melanoma anti-tumor therapy remains a challenge. SiRNA-based therapies provide a powerful means, but limitations remain in its pharmaceutical applications owing to the lack of highly efficient delivery systems. In this study, to improve the siRNA delivery efficiency of chitooligosaccharide (COS), phenylboronic acid (PBA)-modified COS was synthesized and structurally characterized. PBA-modified COS was used to deliver survivin-targeted siRNA for melanoma treatment. The siRNA-loaded nanoparticles were prepared by a synergetic assembly of electrostatic complexation and chemical cross-linking. The particle size and zeta potential were characterized by dynamic light scattering, and transmission electron microscopy was utilized to observe the morphology of the nanoparticles. The cellular uptake of nanoparticles on B16F10 cells was studied by flow cytometry and confocal laser scanning microscopy. A luciferase reporter gene assay determined the gene silencing efficiency of different nanoparticles. As a result, the novel nanoparticles remarkably inhibited the proliferation of B16F10 cells in vitro and significantly inhibited the growth and metastasis of melanoma in vivo. In conclusion, PBA-modified COS can serve as a promising carrier for siRNA delivery in the field of anti-tumor therapy.

Ping Sun,1,2 Wei Huang,1,2 Mingji Jin,1,2 Qiming Wang,1,2 Bo Fan,1,2 Lin Kang,1,2 Zhonggao Gao1,2 1State Key Laboratory of Bioactive Substance and Function of Natural Medicines, 2Department of Pharmaceutics, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China Abstract: Nanoparticle-mediated small interfering RNA (siRNA) delivery is a promising therapeutic strategy in various cancers. However, it is difficult to deliver degradative siRNA to tumor tissue, and thus a safe and efficient vector for siRNA delivery is essential for cancer therapy. In this study, poly(ethylene glycol)-modified chitosan (PEG-CS) was synthesized successfully for delivering nucleic acid drug. We deemed that PEGylated CS could improve its solubility by forming a stable siRNA loaded in nanoparticles, and enhancing transfection efficiency of siRNA-loaded CS nanoparticles in cancer cell line. The research results showed that siRNA loaded in PEGylated CS (PEG-CS/siRNA) nanoparticles with smaller particle size had superior structural stability in the physical environment compared to CS nanoparticles. The data of in vitro antitumor activity revealed that 4T1 tumor cell growth was significantly inhibited and cellular uptake of PEG-CS/siRNA nanoparticles in 4T1 cells was dramatically enhanced compared to naked siRNA groups. The results from flow cytometry and confocal laser scanning microscopy showed that PEG-CS/siRNA nanoparticles were more easily taken up than naked siRNA. Importantly, PEG-CS/siRNA nanoparticles significantly reduced the growth of xenograft tumors of 4T1 cells in vivo. It has been demonstrated that the PEG-CS is a safe and efficient vector for siRNA delivery, and it can effectively reduce tumor growth and prevent metastasis. Keywords: PEGylated chitosan, non-viral vector, siRNA delivery, breast anti-tumor therapy

The aim of the present study was mainly to assess the advantage of docetaxel-loaded PEG-albumin nanoparticles (PEG-DANPs) against non-small cell lung cancer (NSCLC) compared with the commercial product of docetaxel (Aisu®) and docetaxel-albumin nanoparticles (DANPs). We made systematic assessments on these three drugs against NSCLC both in vitro and in vivo. Based on our experiments, PEG-DANPs showed a dose- and time-dependent efficacy in the in vitro cytotoxicity studies; the tumors growth and the metastases in the livers of NSCLC-bearing nude mice in vivo were reduced dmarkedly by PEG-DANPs, and the PEG-DANP-treated mice had a minimum of weight loss; furthermore, the mice which were treated with PEG-DANPs can survive longer than the other groups. In conclusion, the PEG-DANPs have the lowest side-effects, and the highest antitumor and metastases activity of the three drugs, and it may provide an alternative to patients with NSCLC.

The present study mainly compared the effect of docetaxel-albumin nanoparticles (DANPs) and docetaxel-loaded PEG-albumin nanoparticles (PEG-DANPs) against non-small cell lung cancer (NSCLC). We made systematic assessments on these three drugs against NSCLC both in vitro and in vivo. With the purpose of eliminating side-effects of the commercial formulation (Tween-80) and prolonging the blood circulation time, we used emulsion-evaporation cross-link method to prepare DANPs and PEG-DANPs. The DANPs had an average particle size of 163.4 ± 3.76 nm, a zeta potential of -19.4 ± 0.18 mV, a polydispersity index of 0.143 ± 0.03, a drug loading of 8.71 ± 0.98%, and an encapsulation efficiency of 93.58 ± 0.86%; the average particle size of PEG-DANPs is 169.19 ± 2.36 nm, zeta potential is -18.2 ± 0.21 mV, with a polydispersity index of 1.56 ± 0.05, a drug loading of 8.72 ± 1.05% and an encapsulation efficiency of 95.4 ± 5.5%. PEG-DANPs showed a dose- and time-dependent efficacy in cytotoxicity studies in vitro; the hemolysis test indicated that PEG-DANPs had less hemocytolysis than Aisu® and DANPs; in addition, a more prolonged circulation time and sustained in vitro release behavior were observed in the PEG-DANPs compared with Aisu® and DANPs; the cellular uptake test in vitro demonstrated that PEG-DANPs could be absorbed easier into the nucleus; furthermore, the tumor growth of NSCLC-bearing nude mice in vivo was reduced the most by PEG-DANPs. In conclusion, the PEG-DANPs have the lowest side-effects, the highest antitumor activity with the longest blood circulation time of the three drugs, and it will provide an alternative to patients with NSCLC.

Recently, survivin has been attracting great attention because it plays an important role in inhibiting the apoptosis process of tumor cells. Down-regulating the expression of survivin gene by small interfering RNA (siRNA) offers a promising method for anti-tumor therapy. However, lack of appropriate siRNA delivery vector has significantly hindered the successful application of survivin-targeted siRNA in anti-tumor therapy. The purpose of this study was to use polysaccharide vector TAT-g-CS we synthesized to deliver functional siRNA and evaluate its in vivo anti-tumor activity. TAT-g-CS vector was firstly synthesized and well structurally characterized. MTT assay showed that TAT-g-CS vector exhibited good biocompatibility. TAT-g-CS complexed with siRNA offering nanoparticles with an average particle size of 212.2 nm and a polydispersity index of 0.121, and the zeta potential of the nanoparticles was +18.58 mV. Results from reporter gene assay suggested that luciferase-targeted siRNA when delivered by TAT-g-CS could down-regulate the expression of luciferase gene with 75.3% reduction. Most importantly, we use siRNA(Sur) targeting survivin gene to assess the in vitro and in vivo delivery capacity of TAT-g-CS and its anti-tumor effects. Our results demonstrated that TAT-g-CS/siRNA(Sur) nanoparticles not only strongly inhibited the in vitro proliferation of 4T1-Luc tumor cells via inducing cell apoptosis, but also effectively inhibited the in vivo growth and metastasis of malignant breast tumor, which suggested that TAT-g-CS/siRNA nanoparticle was a highly efficient non-viral system for siRNA delivery, especially for anti-tumor therapy based on siRNA therapeutics.

Li-qing Chen, Wei Huang, Zhong-gao Gao, Wei-shuo Fang, Ming-ji Jin State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China Abstract: Lx2-32c is a novel taxane derivative with a strong antitumor activity. In this study, we developed Lx2-32c–loaded polymeric micelles (Lx2-32c-PMs) with small size and investigated their antitumor efficacy against tumor growth and metastasis on 4T1 murine breast cancer cell line with Cremophor EL–based Lx2-32c solution as the control. In this study, copolymer monomethoxy polyethylene glycol2000–polylactide1300 was used to prepare Lx2-32c-PMs by film hydration method, and their physicochemical properties were characterized as well, according to morphology, particle size, zeta potential, in vitro drug release, and reconstitution stability. Under confocal laser scanning microscopy, it was observed that Lx2-32c-PMs could be effectively taken up by 4T1 cells in a time-dependent manner. Cell Counting Kit-8 assay showed that the IC50 of Lx2-32c-PMs was 0.3827 nM. Meanwhile, Lx2-32c-PMs had better ability to promote apoptosis and induce G2/M cycle block and polyploidy formation, compared with Lx2-32c solution. More importantly, in vivo animal studies showed that compared to Lx2-32c solution, Lx2-32c-PMs possessed better ability not only to effectively inhibit the tumor growth, but also to significantly suppress spontaneous and postoperative metastasis to distant organs in 4T1 orthotopic tumor-bearing mice. Consequently, Lx2-32c-PMs have significantly prolonged the survival lifetime of tumor-bearing mice. Thus, our study reveals that Lx2-32c-PMs had favorable antitumor activity and exhibited a good prospect for application in the field of antitumor therapy. Keywords: Lx2-32c, micelles, small size, 4T1 mammary carcinoma, tumor therapy, metastasis

The second generation of pH-sensitive micelles composed of poly(l-histidine-co-l-phenlyalanine(16 mol %))(MW:5K)-b-PEG(MW:2K) and poly(l-lactic acid)(MW:3K)-b-PEG(MW:2K)-folate (80/20 wt/wt %) was previously optimized by physicochemical and in vitro tests for both folate receptor and early endosomal pH targeting (pH approximately 6.0). In this study, the therapeutic efficacy of the doxorubicin (DOX)-loaded micelles (DOX loading content: 20 wt %) was evaluated using in vivo tests. Multidrug-resistant (MDR) ovarian tumor-xenografted mouse models were employed. The skin-fold dorsal window chamber model was applied for visualization of extravasation and drug retention for the initial one hour after iv injection. Noninvasive imaging followed, providing evidence of drug accumulation in the tumor after the first hour. The biodistribution study further supported the long circulation of the drug carrier, tumor-selective accumulation and intracellular drug delivery. Comprehensive tumor growth inhibition experiments examined the collective efficacy of the pH-sensitive micelles. The micelle formulation effectively suppressed the growth of existing MDR tumors in mice for at least 50 days by three iv injections at a 3-day interval at a dose of 10 mg of DOX/kg. The body weight of the animals treated with the test micelle formulation gradually increased over the experimental time period, rather than decreasing. The micelle formulation was superior to its first generation, which targeted pH 6.8 and folate receptor.

A new class of multifunctional nanoparticles that combine properties of polymeric drug carriers, ultrasound imaging contrast agents, and enhancers of ultrasound-mediated drug delivery has been developed. At room temperature, the developed systems comprise perfluorocarbon nanodroplets stabilized by the walls made of biodegradable block copolymers. Upon heating to physiological temperatures, the nanodroplets convert into nano/microbubbles. The phase state of the systems and bubble size may be controlled by the copolymer/perfluorocarbon volume ratio. Upon intravenous injections, a long-lasting, strong and selective ultrasound contrast is observed in the tumor volume indicating nanobubble extravasation through the defective tumor microvasculature, suggesting their coalescence into larger, highly echogenic microbubbles in the tumor tissue. Under the action of tumor-directed ultrasound, microbubbles cavitate and collapse resulting in a release of the encapsulated drug and dramatically enhanced intracellular drug uptake by the tumor cells. This effect is tumor-selective; no accumulation of echogenic microbubbles is observed in other organs. Effective chemotherapy of the MDA MB231 breast cancer tumors has been achieved using this technique.

As an alternative to cell specific cancer targeting strategies (which are often afflicted with the heterogeneity of cancer cells as with most biological systems), a novel polymeric micelle constitute of two block copolymers of poly(L-lactic acid)-b-poly(ethylene glycol)-b-poly(L-histidine)-TAT (transactivator of transcription) and poly(L-histidine)-b-poly(ethylene glycol) was developed. The micelle formed via the dialysis method was approximately 95 nm in diameter and contained 15 wt.% of doxorubicin (DOX) by weight. The micelle surface hides TAT during circulation, which has the strong capability to translocate the micelle into cells, and exposes TAT at a slightly acidic tumor extracellular pH to facilitate the internalization process. The micelle core was engineered for disintegration in early endosomal pH of tumor cells, quickly releasing DOX. The ionization process of the block copolymers and ionized polymers assisted in disrupting the endosomal membrane. This processes permitted high DOX concentrations in the cytosol and its target site of the nucleus, thus increasing DOX potency in various wild and multidrug resistant (MDR) cell lines (3.8-8.8 times lower IC50 than free DOX, depending on cell line). When tested with the xenografted tumors of human ovarian tumor drug-resistant A2780/AD, human breast tumor drug-sensitive MCF-7, human lung tumor A549 and human epidermoid tumor KB in a nude mice model, all tumors significantly regressed in size by three bolus injections at a dose of DOX 10 mg equivalent/kg body per injection of DOX-loaded micelle at three day interval, while minimum weight loss was observed. This approach may replace the need for cell-specific antibodies or targeting ligands, thereby providing a general strategy for solid tumor targeting.

The purpose of this study was to examine the efficacy of a chemotherapeutic drug, doxorubicin (DOX), loaded in pH-sensitive micelles poly(l-histidine) (M(n):5K)-b-PEG (M(n):5K) micelles. The micelles were designed to target the acidic extracellular pH of solid tumors. Studies of pH-dependent cytotoxicity, growth rate of the tumor, pharmacokinetics and biodistribution were conducted. In vitro DOX uptake upon A2780 cells by incubating the cells in a pH 6.8 complete medium at a concentration of 20 microg DOX/ml in the micelle formulation was more than five times that of pH 7.4 condition for initial 20 min. In vivo pharmacokinetic data showed that AUC (area under concentration curve) and half life time (t(1/2)) (plasma half life) of DOX in the pH sensitive micelles increased about 5.8- and 5.2-fold of free DOX in phosphate buffered saline (PBS), respectively. It appeared that DOX in the pH-sensitive micelles preferentially accumulated in the tumor site. The distributions at 12 h post injection in other organs including liver, kidney, spleen, lung and heart were not significantly different from those of DOX in PBS at a 6 mg DOX/kg dose. The in vivo test of anti-tumor activity was performed with human ovarian carcinoma A2780 which was subcutaneously xenografted in female nu/nu athymic mice. The pH-sensitive micelle formulation significantly retarded tumor growth rate without serious body weight loss. The triggered drug release by the reduced tumor pH is believed to be a major mechanism of the observed efficacy after passive accumulation of the micelles by EPR effect. This may have resulted in a local high dose of drug in the tested solid tumor.

The new modality of drug targeting of tumors that we are currently developing is based on drug encapsulation in polymeric micelles, followed by the localized release at the tumor site triggered by focused ultrasound. The rationale behind this approach is that drug encapsulation in micelles decreases systemic concentration of drug, diminishes intracellular drug uptake by normal cells, and provides passive drug targeting of tumors, thus reducing unwanted drug interactions with healthy tissues. Ultrasound irradiation is used to release drug from micelles at the tumor site and to enhance the intracellular drug uptake by tumor cells. An important advantage of ultrasound is that it is noninvasive, can penetrate deep into the interior of the body, can be focused and carefully controlled. Here we describe factors involved in the ultrasound interaction with viable cells in the absence and presence of drug carriers and anti-cancer drugs. We present in vivo effects of 1 MHz ultrasound on drug biodistribution, intratumoral distribution, and survival rates of immuno-compromised athymic nu/nu mice bearing ovarian carcinoma tumors.

Micelles prepared from polyethylene glycols of various lengths conjugated with phosphatidylethanolamine (PEG-PE) were loaded with various poorly soluble anticancer agents. PEG-PE micelles selectively accumulated in Lewis lung carcinoma (LLC) tumors implanted in mice. Modification of the micelles with tumor specific antibodies further enhanced the efficiency of tumor accumulation.

To prepare immunomicelles, new targeted carriers for poorly soluble pharmaceuticals, a procedure has been developed to chemically attach mAbs to reactive groups incorporated into the corona of polymeric micelles made of polyethylene glycol–phosphatidylethanolamine conjugates. Micelle-attached antibodies retained their ability to specifically interact with their antigens. Immunomicelles with attached antitumor mAb 2C5 effectively recognized and bound various cancer cells in vitro and showed an increased accumulation in experimental tumors in mice when compared with nontargeted micelles. Intravenous administration of tumor-specific 2C5 immunomicelles loaded with a sparingly soluble anticancer agent, taxol, into experimental mice bearing Lewis lung carcinoma resulted in an increased accumulation of taxol in the tumor compared with free taxol or taxol in nontargeted micelles and in enhanced tumor growth inhibition. This family of pharmaceutical carriers can be used for the solubilization and enhanced delivery of poorly soluble drugs to various pathological sites in the body.

Pancreatic ductal adenocarcinoma (PDAC) elicits a dense stromal response that blocks vascular access because of pericyte coverage of vascular fenestrations. In this way, the PDAC stroma contributes to chemotherapy resistance, and the small-sized nanocarrier loaded with platinum has been adopted to address this problem which is not suitable for loading docetaxel (DTX). In the present study, we used the poly(d,l-lactide)-b-polyethylene glycol-methoxy (mPEG-b-PDLLA) to encapsulate DTX and got a small-sized polymeric micelle (SPM), meanwhile we functionalized the SPM’s surface with TAT peptide (TAT-PM) for a higher permeability. The diameters of both SPM and TAT-PM were in the range of 15–26 nm. In vitro experiments demonstrated that TAT-PM inhibited Capan-2 Luc PDAC cells growth more efficiently and induced more apoptosis compared to SPM and Duopafei. The in vivo therapeutic efficiencies of SPM and TAT-PM compared to free DTX was investigated on the orthotopic transplantation model of Capan-2 Luc. SPM exerted better therapeutic efficiency than free DTX, however, TAT-PM didn’t outperformed SPM. Overall, these results disclosed that SPM could represent a new therapeutic approach against pancreatic cancer, but its permeability to PDAC was not the only decisive factor.

Shan Liu,1,2 Wei Huang,1,2 Ming-Ji Jin,1,2 Qi-Ming Wang,1,2 Gan-Lin Zhang,3 Xiao-Min Wang,3 Shuai Shao,1,2 Zhong-Gao Gao1,2 1State Key Laboratory of Bioactive Substance and Function of Natural Medicines, 2Department of Pharmaceutics, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China; 3Beijing Hospital of Traditional Chinese Medicine affiliated to Capital Medical University, Beijing, People’s Republic of China Abstract: To our knowledge, the mechanism underlying the high transfection efficiency of ­alkylated low-molecular-weight polyethylenimine (PEI) is not yet well understood. In this work, we grafted branched PEI (molecular weight of 1,800 Da; bPEI1800) with lauryl chains (C12), and found that bPEI1800-C12was structurally similar to gemini surfactant and could similarly assemble into micelle-like particles. Stability, cellular uptake, and lysosome escape ability of bPEI1800-C12/DNA polyplexes were all greatly enhanced after C12grafting. bPEI1800-C12/DNA polyplexes exhibited significantly higher transfection efficiency than Lipofectamine™ 2000in the presence of serum. Bioluminescence imaging showed that systemic injection of bPEI1800-C12/DNA polyplexes resulted in intensive luciferase expression in vivo and bioluminescence signals that could be detected even in the head. Altogether, the high transfection efficacy of bPEI1800-C12was because bPEI1800-C12, being an analog of gemini surfactant, facilitated lysosome escape and induced the coil–globule transition of DNA to assemble into a highly organized micelle-like structure that showed high stability. Keywords: self-organization, alkylation, luciferase, bioluminescence imaging

The bioactive polymer poly( l -glutamic acid) n - b -poly( d , l -lactic acid) m was synthesized and used to form doxorubicin-loaded hybrid polymeric micelles to treat melanoma. These polymers exhibited pH-responsive changes in conformation, which controlled the diverse functionalities of the micelles. During circulation, poly( l -glutamic acid) n - b -poly( d , l -lactic acid) m protected Tat peptides on the micelles from proteolysis. Under tumor-acidic conditions, polymers with shorter poly( l -glutamic acid) blocks underwent a conformational change to form channels that accelerated the release of doxorubicin. The conformational change also exposed the Tat peptides to tumor cells, thereby promoting cellular internalization of the micelles. Enhanced cellular uptake of the micelles induced significant apoptosis of A375 melanoma cells in tumor-acidic conditions. In vivo studies demonstrated that the micelles with shorter poly( l -glutamic acid) blocks could effectively accumulate in tumor tissues, suppress tumor growth and help maintain the body weight of tumor-bearing mice. However, micelles with longer poly( l -glutamic acid) blocks did not undergo a conformational change under acidic conditions and performed poorly in both in vitro and in vivo evaluations. Our work provides a strategy for applying bioactive polymers to the rational construction of pH-responsive delivery systems for solid tumors and lends insight into possible conformational effects on the bioactivity of drug carriers.

Non-viral gene delivery carriers were prepared by using DNA/polyethylenimine/polymethacrylic acid (DPP) polyplexes and its extravasation from tumor blood vessel was evaluated with mouse dorsal skin fold window chamber model. The DNA/PEI (DP) complex with a ratio of N to P (10/1) was coated with polymethacrylic acid, and the ratio of PMA to DNA complex in DNA/PEI/PMA (DPP) polyplex was fixed 0.03 (w/w). The surface charges of the DP and DPP polyplex were positive 26 and 15, respectively. The size of DP and DPP polyplex were 161 nm and 195 nm. The transfection efficiencies in HepG2 cells were about 30-fold and 20-fold higher than that in HeLa and L/C cells in the presence of 50% serum, respectively. The DPP polyplex showed a reduced erythrocyte aggregation activity and a decreased cytotoxicity in cancer cells. After being incubated 30 min, Fluorescently labelled DPP polyplex uptaken by cancer cells decreased, compared with DP by measuring flowcytometry. DPP polyplex penetrating through tumor blood vessel appeared fast and stayed longer in tumour interstitial, this fact was observed from mouse dorsal skin fold window chamber model.

Background Drug delivery in polymeric micelles combined with tumor irradiation by ultrasound results in effective drug targeting, but this technique requires prior tumor imaging. A technology that combined ultrasound imaging with ultrasound-mediated nanoparticle-based targeted chemotherapy could therefore have important applications in cancer treatment. Methods Mixtures of drug-loaded polymeric micelles and perfluoropentane (PFP) nano/microbubbles stabilized by the same biodegradable block copolymer were prepared. Size distribution of nanoparticles was measured by dynamic light scattering. Cavitation activity (oscillation, growth, and collapse of microbubbles) under ultrasound was assessed based on the changes in micelle/microbubble volume ratios. The effect of the nano/microbubbles on the ultrasound-mediated cellular uptake of doxorubicin (Dox) in MDA MB231 breast tumors in vitro and in vivo (in mice bearing xenograft tumors) was determined by flow cytometry. Statistical tests were two-sided. Results Phase state and nanoparticle sizes were sensitive to the copolymer/perfluorocarbon volume ratio. At physiologic temperatures, nanodroplets converted into nano/microbubbles. Doxorubicin was localized in the microbubble walls formed by the block copolymer. Upon intravenous injection into mice, Dox-loaded micelles and nanobubbles extravasated selectively into the tumor interstitium, where the nanobubbles coalesced to produce microbubbles with a strong, durable ultrasound contrast. Doxorubicin was strongly retained in the microbubbles but released in response to therapeutic ultrasound. Microbubbles cavitated under the action of tumor-directed ultrasound, which enhanced intracellular Dox uptake by tumor cells in vitro to a statistically significant extent relative to that observed with unsonicated microbubbles (drug uptake ratio = 4.60; 95% confidence interval [CI] = 1.70 to 12.47; P = .017) and unsonicated micelles (drug uptake ratio = 7.97; 95% CI = 3.72 to 17.08; P = .0032) and resulted in tumor regression in the mouse model. Conclusions Multifunctional nanoparticles that are tumor-targeted drug carriers, long-lasting ultrasound contrast agents, and enhancers of ultrasound-mediated drug delivery have been developed and deserve further exploration as cancer therapeutics.

Cancer chemotherapy is often complicated by toxic side effects of anticancer drugs. We are developing a new modality of tumor chemotherapy aimed at circumventing side effects of treatment via drug targeting to tumors. The technique is based on drug encapsulation in polymeric micelles followed by a controlled drug release at a target site triggered by ultrasonic irradiation. The encouraging in vitro results of previous years warranted animal experiments for verification of the in vivo feasibility of the proposed technique. We report here on the effect of ultrasound on a biodistribution of a micellar drug carrier (fluorescently labeled Pluronic micelles) in ovarian cancer-bearing nu/nu mice. The degree of carrier accumulation in the cells of various organs was characterized by flow cytometry. Polymeric micelles were formed in either pure Pluronic P-105 solutions (unstabilized micelles) or 1:1 (weight) mixtures of Pluronic P-105 with PEG-diacylphospholipid (stabilized micelles). Intraperitoneal (ip) and intravenous (iv) injections were compared. The data showed that a 30 s ultrasonic irradiation by 1 or 3 MHz ultrasound applied locally to the tumor significantly enhanced accumulation of Pluronic in the tumor cells, which was observed for both ip and iv injections and for unstabilized and stabilized micelles. The data indicated targeting of Pluronic micelles to the tumors; the degree of targeting was enhanced by a local tumor sonication.

The results of a comprehensive in vivo study of a novel tumor-targeting modality are reported. The technique utilized in this study is based on the encapsulation of the chemotherapeutic agent within polymeric micelles in combination with a local ultrasonic irradiation of the tumor. A doxorubicin (DOX) biodistribution, a yield of the internal tumors and a growth rate of the subcutaneous (s.c.) tumors was compared for molecularly dissolved and micellar-encapsulated DOX. This was done with and without tumor sonication, using an ovarian carcinoma tumor model in nu/nu mice. Pure and mixed Pluronic P-105, PEG2000-diacylphospholipid, and poly(ethylene glycol)-co-poly(beta-benzyl-L-aspartate) micelles were used as drug carriers. DOX intracellular uptake was characterized by flow cytometry. A local ultrasonic irradiation of the tumor resulted in a substantially increased drug accumulation in the tumor cells. The effect of the ultrasound was dependent on the time between ultrasound application and drug injection. Ultrasound did not enhance micelle extravasation; the ultrasonic enhancement of drug internalization by the tumor cells required a preliminary passive drug accumulation in the tumor interstitium. Due to the ultrasound-enhanced drug intracellular uptake and cell killing, the yield of intraperitoneal (i.p.) ovarian carcinoma tumors decreased from 70% for DOX dissolved in PBS (positive control) to 36% for the same concentration of DOX encapsulated in Pluronic micelles combined with a 30-s sonication of the abdominal region of a mouse (3 mg/kg DOX, i.p. injection 1 day after inoculation, n>or=10). For s.c. tumors, micellar delivery combined with localized ultrasonic tumor irradiation resulted in a substantial decrease of the tumor growth rates compared to a positive control (3 mg/kg DOX, i.v. injections, n=7, p

Mixed micelles were prepared from poly(ethyleneglycol)-distearyl phosphoethanolamine (PEG 2000 -PE) and egg phosphatidylcholine. The micelles were covalently modified with the nucleosome-specific monoclonal antibody 2C5 known to recognize and bind a variety of tumor cells via their surface-bound nucleosomes. Covalent attachment of 2C5 antibody was performed via a micelle-incorporated PEG-PE with the distal terminus of the PEG block activated with p -nitrophenylcarbonyl group (pNP-PEG-PE). Micelle surface-attached 2C5 antibody maintained its specific activity. 2C5-targeted immunomicelles were able to carry more than 3 wt% of taxol. Taxol-loaded immunomicelles specifically recognized tumor cell lines of several types. The cytotoxicity of 2C5-targeted taxol-loaded immunomicelles in a cell culture model was much higher when compared with free taxol or taxol in non-targeted micelles.

The purpose of this work is to study the potential of micelles prepared from amphiphilic polyethelene glycol/phosphatidylethanolamine (PEG-PE) conjugates as a particulate drug delivery system capable of accumulation in tumors via the enhanced permeability and retention (EPR) effect.Micelles were prepared from PEGs of different molecular lengths conjugated with PE. The micelles were characterized by fluorescence-based critical micellization concentration (CMC) measure ments, dynamic light scattering, and HPLC. Blood clearance an tumor accumulation of 111In-labeled micelles were studied in mic with subcutaneously established Lewis lung carcinoma (LLC) and EL4 T lymphoma (EL4) tumors.Various versions of PEG-PE conjugates with PEG blocks ranging from 750 to 5000 Da formed very stable low CMC micelles at all concentrations down to 10(-5) M. The size of the micelles varie between 7 and 35 nm depending on the length of the PEG block. Micelles remained intact after prolonged incubation with the blood serum. Upon intravenous administration into mice, the micelles demonstrated circulation longevity, and they efficiently and selectively accumulated in both subcutaneous Lewis lung carcinoma and EL4 T lymphoma tumors.PEG-PE conjugates form very stable, long-circulating micelles. These micelles efficiently accumulate in tumors in vivo an may potentially be used as a tumor-specific delivery system for poorly soluble anticancer drugs.