Preparation of poly(glutamic acid) shielding micelles self-assembled from polylysine-b-polyphenylalanine for gene and drug codelivery

A novel amphiphilic cationic block copolymer polylysine-b-polyphenylalanine(PLL-b-PPhe) was synthesized and self-assembled into micelles in aqueous solution,then shielded with poly(glutamic acid)(marked as PG/PLL-b-PPhe) to codeliver gene and drug for combination cancer therapy.Here,doxorubicin(DOX) was selected to be loaded into PLL-b-PPhe micelles and the drug loading efficiency was 8.0%.The drug release studies revealed that the PLL-b-PPhe micelles were pH sensitive and the released DOX could reach to 53.0%,65.0%,72.0% at pH 7.4,6.8 and 5.0,respectively.In order to reduce positive charge and cytotoxicity of PLL-b-PPhe micelles,PG was used as shelding,simultaneously condensed with Bcl2 siRNA to form gene carrier system.Compared with PEI,PG/PLL-b-PPhe had excellent gene transfection efficiency,especially when the molar ratio of PLL to PPhe was 30:60 and the mixed mass ratio of PLL-b-PPhe to gene was 5:1.More importantly,DOX and Bcl2 siRNA gene codelivery system displayed remarkable cytotoxicity against B16 F10 cells.Confocal laser scanning microscopy(CLSM) and flow cytometry were used to characterize endocytosis of the codelivery system,and confirmed that both DOX and Bcl2 siRNA had been endocytosed into B16 F10 cells.The above results indicated that gene and drug codelivery was a promising strategy in future cancer therapy.     

Integrated Hollow Mesoporous Silica Nanoparticles for Target Drug/siRNA Co‐Delivery

A hollow mesoporous silica nanoparticle (HMSNP) based drug/siRNA co‐delivery system was designed and fabricated, aiming at overcoming multidrug resistance (MDR) in cancer cells for targeted cancer therapy. The as‐prepared HMSNPs have perpendicular nanochannels connecting to the internal hollow cores, thereby facilitating drug loading and release. The extra volume of the hollow core enhances the drug loading capacity by two folds as compared with conventional mesoporous silica nanoparticles (MSNPs). Folic acid conjugated polyethyleneimine (PEI‐FA) was coated on the HMSNP surfaces under neutral conditions through electrostatic interactions between the partially charged amino groups of PEI‐FA and the phosphate groups on the HMSNP surfaces, blocking the mesopores and preventing the loaded drugs from leakage. Folic acid acts as the targeting ligand that enables the co‐delivery system to selectively bind with and enter into the target cancer cells. PEI‐FA‐coated HMSNPs show enhanced siRNA binding capability on account of electrostatic interactions between the amino groups of PEI‐FA and siRNA, as compared with that of MSNPs. The electrostatic interactions provide the feasibility of pH‐controlled release. In vitro pH‐responsive drug/siRNA co‐delivery experiments were conducted on HeLa cell lines with high folic acid receptor expression and MCF‐7 cell lines with low folic acid receptor expression for comparison, showing effective target delivery to the HeLa cells through folic acid receptor meditated cellular endocytosis. The pH‐responsive intracellular drug/siRNA release greatly minimizes the prerelease and possible side effects of the delivery system. By simultaneously delivering both doxorubicin (Dox) and siRNA against the Bcl‐2 protein into the HeLa cells, the expression of the anti‐apoptotic protein Bcl‐2 was successfully suppressed, leading to an enhanced therapeutic efficacy. Thus, the present multifunctional nanoparticles show promising potentials for controlled and targeted drug and gene co‐delivery in cancer treatment.     

A Biodegradable Polymersome Containing Bcl‐xL siRNA and Doxorubicin as a Dual Delivery Vehicle for a Synergistic Anticancer Effect

Combined cancer treatment via co‐delivery of siRNAs and an anticancer drug can be a promising strategy due to the synergistic effect of simultaneously minimizing gene/drug administration. In this study, Bcl‐xL siRNA and doxorubicin (DOX) are encapsulated into designed methoxy‐poly(ethylene glycol)‐block‐poly(D ,L ‐lactic acid) (mPEG‐b‐PLA) block copolymer polymersomes (PSomes). A study of the cytotoxicity of Bcl‐xL siRNA and DOX co‐encapsulated PSomes (CPSomes) shows more inhibited proliferation of MKN‐45 and MKN‐28 human gastric cancer cell lines than only gene‐ and drug‐loaded ones. Consequently, these results demonstrate that co‐delivery of genes and drugs using PSomes results in a synergistic efficacy and indicates the potential of PSomes as efficient nanocarriers for combined cancer therapy.

     

Antitumor immunity induced by tumor cells engineered to express a membrane-bound form of IL-2

Transduction of cytokine gene into tumor cells is a promising method of tumor therapy, but the value is limited by accompanying side effects. To focus antitumor immune response to tumor antigen-specific CTL, we developed an antitumor vaccine by transfecting modified IL-2 gene in a membrane-bound form (mbIL-2) into B16F10 melanoma cells. The mbIL-2 clone showed reduced tumorigenicity and metastatic ability, and inhibited metastasis and prolonged the survival of mice against B16F10 cells. The inhibition of B16F10 metastasis by mbIL-2 was accompanied by the increment of CD8(+) T cells. The metastasis of mbIL-2 clone was significantly increased in the CD8(+) T cell-depleted mice, but not in CD4(+) T cell depleted mice. Spleen cells immunized with the mbIL-2 clone showed higher CTL activity towards B16F10 cells than those immunized with control cells. The size of CD8(+) T cell population in the lung of mice injected with the mbIL-2 clone was markedly greater than that of mice injected with B16F10 cells, but there was no detectible change in CD4(+) and CD8(+) T cell populations of lymph nodes and spleen. These results suggest that when the mbIL-2 clone is introduced into the blood stream, it migrates mainly to lung and activates CD8(+) T cells in situ, possibly by direct priming. Such a tumor vaccine may ameliorate the toxic side effects encountered with conventional cytokine gene therapy.     

Polymeric Nanoparticles Induce NLRP3 Inflammasome Activation and Promote Breast Cancer Metastasis

Polymeric nanoparticles gain enormous interests in cancer therapy. Polyethylenimine (PEI) 25 kD is well known for its high transfection efficiency and cytotoxicity. PEI‐CyD (PC) was previously synthesized by conjugating low molecular PEI (M _w 600) with β‐cyclodextrin (β‐CyD), which is shown to induce lower cytotoxicity than PEI 25 kD. In the current study, the in vivo immune response of branched PEI 25 kD and PC is investigated. Compared to PC/pDNA, exposure of PEI 25kD/pDNA induces higher level of immune‐stimulation evidenced by the increased spleen weight, phagocytic capacity of peritoneal macrophage, and proinflammatory cytokines in serum and liver. Importantly, administration of PEI 25 kD can greatly promote breast cancer metastasis in liver and lung tissues, which correlates with its ability to induce high oxidative stress and NLRP3‐inflammasome activation. These results suggest that polymeric nanocarriers have the potential to induce immune‐stimulation and cancer metastasis, which may affect their efficiency for cancer therapy.     

Polymeric prodrug for bio-controllable gene and drug co-delivery

A polymeric polyethylenimine (PEI)-based prodrug of anticancer doxorubicin (DOX) (PEI-hyd-DOX) was designed by attaching DOX to PEI via an acid-labile hydrazone bond, for the achievement of biocontrollable gene and drug co-delivery in response to the intracellular acid microenvironments in the late endosome/lysosome compartments. The cytotoxicity of PEI-hyd-DOX was evaluated by the MTT assay and the cellular uptake was monitored using confocal laser scanning microscopy. The polymeric prodrug can respond with a high sensitivity to the specific acid condition inside cells, thus permitting the precise biocontrol over intracellular drug liberation with high drug efficacy. The chemical attachment of drug molecules also led to the relatively reduced toxicity and the enhanced transfection efficiency compared with parent PEI. The resulting data adumbrated the potential of PEI-hyd-DOX to co-deliver DOX and therapeutic gene for the combination of chemotherapy and gene therapy.     

Enhanced antitumor chemo-immunotherapy by local co-delivery of chemotherapeutics,immune checkpoint blocking antibody and IDO inhibitor using an injectable polypeptide hydrogel

The efficiency of antitumor immunotherapy is usually limited by the immunosuppressive tumor microenvironment (TME). In this study, we developed a chemo-immunotherapy strategy that is able to improve the immunosuppressive TME for enhancing the antitumor efficacy. The chemo-immunotherapy was achieved by the topical co-delivery of a chemotherapeutic drug, Doxorubicin (DOX), an immune checkpoint blocking antibody targeting programmed cell death protein 1 (aPD-1), and an indoleamine-2,3-dioxygenase (IDO) inhibitor, 1-methyl-d -tryptophan (d -1MT) by using a thermosensitive polypeptide hydrogel. It was revealed that the sustained DOX release from the hydrogel caused the immunogenic cell death (ICD) of B16F10 cells in vitro, and the tumor cell lysates subsequently promoted the activation of dendritic cells (DCs). After intratumoral injection into B16F10 melanoma-bearing mice, the DOX/aPD-1/D-1MT co-loaded hydrogel exhibited enhanced tumor inhibition efficacy and prolonged animal survival time, compared to the DOX/aPD-1/D-1MT mixed solution, DOX-loaded hydrogel or DOX/aPD-1 co-loaded hydrogel. The improvement of immunosuppressive TME and enhancement of antitumor immune response after the local chemo-immunotherapy were demonstrated by the augmented activation of DCs and increased infiltration of CD8⁺ and CD4⁺ T cells, as well as enhanced secretion of pro-inflammatory cytokines. Therefore, the hydrogel-based local chemo-immunotherapy system holds great potential for effective antitumor treatment.     

Systemic Delivery of Bc12‐Targeting siRNA by DNA Nanoparticles Suppresses Cancer Cell Growth

Short interfering RNA (siRNA) is a promising molecular tool for cancer therapy, but its clinical success is limited by the lack of robust in vivo delivery systems. Rationally designed DNA nanoparticles (DNPs) have emerged as facile delivery vehicles because their physicochemical properties can be precisely controlled. Nonetheless, few studies have used DNPs to deliver siRNAs in vivo, and none has demonstrated therapeutic efficacy. Herein, we constructed a number of DNPs of rectangular and tubular shapes with varied dimensions using the modular DNA brick method for the systemic delivery of siRNA that targets anti‐apoptotic protein Bcl2. The siRNA delivered by the DNPs inhibited cell growth both in vitro and in vivo, which suppressed tumor growth in a xenograft model that specifically correlated with Bcl2 depletion. This study suggests that DNPs are effective tools for the systemic delivery of therapeutic siRNA and have great potential for further clinical translation.     

Doxorubicin-loaded PLGA microparticles with internal pores for long-acting release in pulmonary tumor inhalation treatment

Doxorubicin(DOX) loaded poly(lactic-co-glycolic acid)(PLGA) microparticles with internal pores(MP-D) were developed for long-acting release in pulmonary inhalation treatment. The PLGA microparticles exhibited favorable aerodynamic properties for pulmonary delivery. In vitro drug release profile suggested that MP-D have the advantage of long-term maintenance of drug concentrations. MTT assay demonstrated the in vitro anti-tumor efficiency of the DOX loaded PLGA microparticles. Furthermore, melanoma lung metastasis model was established to determine the in vivo antitumor efficiency. The mice treated with MP-D showed significantly fewer lesions than the untreated ones. The survival analysis indicated that MP-D prolonged the survival time of tumor-bearing mice. These results suggested that DOX loaded PLGA microparticles with internal pores have the potential to be used as long-acting release carriers in clinical lung cancer treatment.     

刺激响应共负载药物/基因微载体的制备

合成了聚乙烯亚胺接枝二茂铁(PEI-Fc)两亲聚合物, 采用水包油法制备包埋疏水性抗癌药阿霉素(DOX)的载药胶束, 并利用胶束表面正电荷的PEI链段有效缔合DNA, 获得尺寸合适、 表面带正电荷的阿霉素与基因共负载微载体. 在磷酸盐(PBS)缓冲溶液中, 共负载微载体能够缓慢释放出DOX. 在硝酸铈铵存在下, 二茂铁从疏水性转变为亲水性, 使载药胶束完全解离, 由于PEI-Fc与DNA之间的静电作用, 使基因超分子组装体稳定存在, 显示出很好的氧化响应特性. 细胞培养结果表明, 表面带正电荷的共负载微载体易被HepG2细胞内吞, 并可转染, 且随着DOX的释放逐渐杀死HepG2肝癌细胞, 为安全稳定、 具有刺激响应的药物与基因共负载微载体的制备提供了可行的途径.     

High-molecular-weight polyethyleneimine conjuncted pluronic for gene transfer agents

In order to enhance the gene delivery efficiency and decrease cytotoxicity of polyplexes, copolymers consisting of branched polyethyleneimine (PEI) 25 kDa grafted with Pluronic (F127, F68, P105) were successfully synthesized using a simple two-step procedure. The copolymers were tested for cytotoxicity and DNA condensation and complexation properties. Their polyplexes with plasmid DNA were characterized in terms of DNA size and surface charge and transfection efficiency. The complex sizes were below 300 nm, which implicated their potential for intracellular delivery. The Pluronic-g-PEI exhibited better condensation and complexation properties than PEI 25 kDa. The cytotoxicity of PEI was strongly reduced after copolymerization. The Pluronic-g-PEI showed lower cytotoxicity in three different cell lines (Hela, MCF-7, and HepG2) than PEI 25 kDa. pGL3-lus was used as a reporter gene, and the transfection efficiency was in vitro measured in HeLa cells. Compared with unmodified PEI 25 kDa Pluronic-g-PEI showed much higher transfection efficiency. These results demonstrate that polyplexes prepared using a combined strategy of surface crosslinking and grafted with Pluronic seem to provide promising properties as stable, high transfection efficiency vectors.     

2-Phenylindole derivatives as anticancer agents: synthesis and screening against murine melanoma,human lung and breast cancer cell lines

Indole derivatives have attractive anticancer properties and may be a future hope for better anticancer drug(s) of low toxicity and high potency. In this paper, syntheses of 2-phenylindole derivatives have been described via Fischer indole synthesis through a one-pot solvent-free method. The synthesized compounds were screened for anticancer potential in vitro against murine melanoma (B16F10), human lung cancer (A549), and human breast cancer (MDA-MB-231) cell lines. The results highlighted that 2-phenylindole derivatives are also promising anticancer agents in case of melanoma and lung cancer along with the breast cancer. Molecular docking analyses with possible targets for melanoma (NEDD4-1) and lung cancer (EGFR) were also performed to understand specific interactions of 2-phenylindole derivatives with the amino acid residues of the receptors.     

Rattle-type hollow CaWO4:Tb(3+)@SiO2 nanocapsules as carriers for drug delivery

Rattle-type hollow nanocapsules are among of the most promising candidates as drug carriers owing to their huge inner space and multifunctional material combination. In this paper, rattle-type hollow CaWO(4):Tb(3+)@SiO(2) nanocapsules with a diameter of 100-110 nm and a wall thickness around 10 nm were fabricated. The hollow silica nanospheres were used as nano-reactors and the luminescent core of CaWO(4):Tb(3+) was post-filled into the nano-reactors by a vacuum nano-casting route combined with a Pechini-type sol-gel method. Subsequently, doxorubicin hydrochloride (DOX), a model of an anti-cancer drug, is loaded into the CaWO(4):Tb(3+)@SiO(2) nanocapsules and their cell cytotoxicity, cancer cell uptake and drug release behavior are investigated in vitro. The prepared multifunctional inorganic nanocapsules show a loading capacity for DOX as high as 124 mg g(-1) and sustained-release properties. The release profile of the drug from DOX-loaded nanocapsules can last over five days. Besides, the blank CaWO(4):Tb(3+)@SiO(2) shows very low cytotoxicity against cancer cell lines (HeLa cell) while the DOX-loaded nanocapsules exhibit relatively high efficiency for killing of HeLa cells. The rapid cancer cell uptake process is observed by confocal laser scanning microscopy. The results indicate that a rattle-type hollow CaWO(4):Tb(3+)@SiO(2) nanocapsule has the potential to be used as drug carrier in therapy. Moreover, it is possible to extend the synthetic strategy in this study to other rattle-type multifunctional composites to meet various demands.     

Virus-Like Particle-Encapsulated Doxorubicin Enters and Kills Murine Tumor Cells Differently from Free Doxorubicin

The use of nanoparticles as chemotherapeutic carriers has been suggested as a way to overcome a range of side effects associated with classical cancer treatment such as poor selectivity and tumor resurgence. Obtaining precise control of the size and shape of therapeutic nanoparticles is crucial to optimize the targeting of tumor sites. In this work, it is shown that a previously developed system of polypeptide encapsulating individual DNA molecules, that forms rod-shaped nanoparticles of precisely controlled aspect ratio, can be loaded with the DNA-intercalating chemotherapeutic drug doxorubicin (DOX). It is characterized the size and shape of the DOX loaded-Virus-Like DNA Particles (DOX-VLDP) and shown that in this system the DOX payload does not leak out. Through in vitro cell studies, it is shown that DOX-VLDP is internalized by melanoma tumor cells (B16F10 cells) in a delayed and endocytosis-dependent way culminating in increased cytotoxicity and selectivity to tumor cells in comparison with free DOX. In addition, it is found that DOX-VLDP trigger apoptosis and autophagy pathways in treated cells. Taken together, the data on the DOX-VLDP nanoparticles shows that they kill cancer cells differently from free DOX.     

In situ vaccination and gene-mediated PD-L1 blockade for enhanced tumor immunotherapy

Despite of the promising achievements of immune checkpoints blockade therapy (ICB) in the clinic, which was often limited by low objective responses and severe side effects. Herein, we explored a synergistic strategy to combine in situ vaccination and gene-mediated anti-PD therapy, which was generated by unmethylated cytosine-phosphate-guanine (CpG) and pshPD-L1 gene co-delivery. PEI worked as the delivery carrier to co-deliver the CpG and pshPD-L1 genes, the formed PDC (PEI/DNA/CpG) nanoparticles were further shielded by aldehyde modified polyethylene glycol (OHC-PEG-CHO) via pH responsive Schiff base reaction for OHC-PEG-CHO-PEI/DNA/CpG nanoparticles (P(PDC) NPs) preparation. All steps could be finished within 30 min. Such simple nanoparticles achieved the synergistic antitumor efficacy in B16F10 tumor-bearing mice, and the amplified T cell responses, together with enhanced NK cells infiltration were observed after the combined treatments. In addition, the pH responsive delivery system reduced the side effects triggered by anti-PD therapy. The facile and effective combination strategy we presented here might provide a novel treatment for tumor inhibition.     

Biocompatible Triplex Ag@SiO2@mTiO2 Core–Shell Nanoparticles for Simultaneous Fluorescence‐SERS Bimodal Imaging and Drug Delivery

Herein, we report the synthesis of biocompatible triplex Ag@SiO₂@mTiO₂ core–shell nanoparticles (NPs) for simultaneous fluorescence‐surface‐enhanced Raman scattering (F‐SERS) bimodal imaging and drug delivery. Stable Raman signals were created by typical SERS tags that were composed of Ag NPs for optical enhancement, a reporter molecule of 4‐mercaptopyridine (4‐Mpy) for a spectroscopic signature, and a silica shell for protection. A further coating of mesoporous titania (mTiO₂) on the SERS tags offered high loading capacity for a fluorescence dye (flavin mononucleotide) and an anti‐cancer drug (doxorubicin (DOX)), thereby endowing the material with fluorescence‐imaging and therapeutic functions. The as‐prepared F‐SERS dots exhibited strong fluorescence when excited by light at 460 nm whilst a stable, characteristic 4‐Mpy SERS signal was detected when the excitation wavelength was changed to longer wavelength (632.8 nm), both in solution and after incorporation inside living cells. Their excellent biocompatibility was demonstrated by low cytotoxicity against MCF‐7 cells, even at a high concentration of 100 μg mL^?1. In vitro cell cytotoxicity confirmed that DOX‐loaded F‐SERS dots had a comparable or even greater therapeutic effect compared with the free drug, owing to the increased cell‐uptake, which was attributed to the possible endocytosis mechanism of the NPs. To the best of our knowledge, this is the first proof‐of‐concept investigation on a multifunctional nanomedicine that possessed a combined capacity for fast and multiplexed F‐SERS labeling as well as drug‐loading for cancer therapy.     

Covalent Modification of Reduced Graphene Oxide by Means of Diazonium Chemistry and Use as a Drug‐Delivery System

Under acidic conditions, reduced graphene oxide (rGO) was functionalized with p‐aminobenzoic acid, which formed the diazonium ions through the diazotization with a wet‐chemical method. Surfactants or stabilizers were not applied during the diazotization. After the functionalized rGO was treated through mild sonication in aqueous solution, these functionalized rGO sheets were less than two layers, which was determined by atomic force microscopy (AFM) imaging. The water solubility of functionalized rGO after the introduction of polyethyleneimine (PEI) was improved significantly; it was followed by covalent binding of folic acid (FA) molecules to the functionalized rGO to allow us to specifically target CBRH7919 cancer cells by using FA as a receptor. The loading and release behaviors of elsinochrome A (EA) and doxorubicin (DOX) on the functionalized rGO sheets were investigated. The EA loading ratio onto rGO‐C₆H₄‐CO‐NH‐PEI‐NH‐CO‐FA (abbreviated rGO‐PEI‐FA, the weight ratio of drug loaded onto rGO‐PEI‐FA) was approximately 45.56 %, and that of DOX was approximately 28.62 %. It was interesting that the drug release from rGO‐PEI‐FA was pH‐ and salt‐dependent. The results of cytotoxicity (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) and flow cytometry (FCM) assays, as well as cell morphology observations) clearly showed that the concentration of rGO‐PEI‐FA as the drug‐delivery composite should be less than 12.5 mg L ^?1. The conjugation of DOX and rGO‐PEI‐FA can enhance the cancer‐cell apoptosis effectively and can also push the cancer cells to the vulnerable G2 phase of the cell cycle, which is most sensitive and susceptible to damage by drugs or radiation.     

Site-specific drug delivery to the lung

Investigations were undertaken to determine whether anti-cancer drugs introduced locally into the lung using bioerodible polyanhydride microspheres as carriers would demonstrate both efficacy and reduced toxicity. BCNU (carmustine) and CDDP (cisplatin), loaded in polyanhydride microspheres, were administered to mice bearing either of two tumors selected for their affinity for the lung: the B16F10 melanoma and the recently established GL26F₄ glioma. Following intravenous inoculation of these tumor cells, the number of metastatic foci formed in the lung follows a predictable time course and can readily be determined. Comparisons were made between the efficacy of microspheres introduced into the lung by intratracheal intubation (IT) and of those administered by intraperitoneal injection (IP). Administration of microspheres loaded either with BCNU or cisplatin reduced the detectable metastatic foci by 25–90% depending on the tumor load, both when administered IP and IT. Toxicity was assessed by flow cytometric analysis of bone marrow cellularity as well as by determination of mortality rates. Intratracheal administration of either cisplatin or BCNU reduced the deleterious systemic effects observed when the drug was administered by IP injection. This was seen at high drug levels, where significant mortality occurred only in animals given drug injections IP; and at lower levels where IP injection led to a reduction of bone marrow blast cells, while IT administration caused no detectable effect on marrow cellularity. Since local delivery of BCNU or cisplatin through bioerodible polyanhydride microspheres induces significantly less systemic damage while demonstrating efficacy equal to or exceeding that of IP injection, this mode of drug delivery warrants further and more detailed exploration. Moreover, the method may be applicable to the treatment of other chronic pulmonary pathologies.