Polymeric nanoparticles for pulmonary protein and DNA delivery

Polymeric nanoparticles (NPs) are promising carriers of biological agents to the lung due to advantages including biocompatibility, ease of surface modification, localized action and reduced systemic toxicity. However, there have been no studies extensively characterizing and comparing the behavior of polymeric NPs for pulmonary protein/DNA delivery both in vitro and in vitro. We screened six polymeric NPs: gelatin, chitosan, alginate, poly(lactic-co-glycolic) acid (PLGA), PLGA–chitosan and PLGA–poly(ethylene glycol) (PEG), for inhalational protein/DNA delivery. All NPs except PLGA–PEG and alginate were <300 nm in size with a bi-phasic core compound release profile. Gelatin, PLGA NPs and PLGA–PEG NPs remained stable in deionized water, serum, saline and simulated lung fluid (Gamble’s solution) over 5 days. PLGA-based NPs and natural polymer NPs exhibited the highest cytocompatibility and dose-dependent in vitro uptake, respectively, by human alveolar type-1 epithelial cells. Based on these profiles, gelatin and PLGA NPs were used to encapsulate plasmid DNA encoding yellow fluorescent protein (YFP) or rhodamine-conjugated erythropoietin (EPO) for inhalational delivery to rats. Following a single inhalation, widespread pulmonary EPO distribution persisted for up to 10 days while increasing YFP expression was observed for at least 7 days for both NPs. The overall results support both PLGA and gelatin NPs as promising carriers for pulmonary protein/DNA delivery.     

Effects of mannose density on in vitro and in vivo cellular uptake and RNAi efficiency of polymeric nanoparticles

To evaluate the effects of mannose density on in vitro and in vivo cellular uptake and RNA interference (RNAi) efficiency of polymeric nanoparticles (NPs) in macrophages, mannose-modified trimethyl chitosan-cysteine (MTC) conjugates with mannose densities of 4%, 13%, and 21% (MTC-4, MTC-13, and MTC-21) were synthesized. Tumor necrosis factor-alpha (TNF-α) siRNA loaded MTC NPs with particle sizes of ∼150 nm exhibited desired structural stability and effectively protected siRNA from enzymatic degradation. Generally, cellular uptake and RNAi efficiency were affected by mannose density. As expected, MTC-21 NPs presented the maximum in vitro uptake and RNAi efficacy in Raw 264.7 cells among all NPs tested. However, MTC-4 NPs exhibited the optimal in vivo uptake by peritoneal exudate cell macrophages (PECs). In the inflammation model of acute hepatic injury, orally delivered MTC-4 and MTC-13 NPs worked better in silencing TNF-α expression and alleviating liver damage than MTC-21 NPs. As for the ulcerative colitis model, MTC-4 NPs outperformed MTC-13 and MTC-21 NPs with respect to TNF-α knockdown and therapeutic efficacy following oral administration. These results highlighted the importance of ligand density in cellular uptake and RNAi efficiency, which could serve as a guideline in the rational design of targeted nanocarriers for anti-inflammation therapy.     

Tumor-specific penetrating peptides-functionalized hyaluronic acid-d-α-tocopheryl succinate based nanoparticles for multi-task delivery to invasive cancers

Poor site-specific delivery and incapable deep-penetration into tumor are the intrinsic limitations to successful chemotherapy. Here, the tumor-homing penetrating peptide tLyP-1-functionalized nanoparticles (tLPTS/HATS NPs), composed of two modularized amphiphilic conjugates of tLyP-1-PEG-TOS (tLPTS) and TOS-grafted hyaluronic acid (HATS), had been fabricated for tumor-targeted delivery of docetaxel (DTX). The prepared tLPTS/HATS NPs had about 110 nm in mean diameter, high drug encapsulation efficiency (93%), and sustained drug release behavior. In vitro studies demonstrated that the tLPTS/HATS NPs exhibited enhanced intracellular delivery and much better anti-invasion ability, cytotoxicity, and apoptosis against both invasive PC-3 and MDA-MB-231 cells as compared to the non-tLyP-1-functionalized HATS NPs. The remarkable penetrability and inhibitory effect on both PC-3 and MDA-MB-231 multicellular tumor spheroids were also identified for the tLPTS/HATS NPs. In vivo biodistribution imaging demonstrated the tLPTS/HATS NPs possessed much more lasting accumulation and extensive distribution throughout tumor regions than the HATS NPs. The higher in vivo therapeutic efficacy with lower systemic toxicity of the tLPTS/HATS NPs was also verified by the PC-3 xenograft model in athymic nude mice. These results suggested that the designed novel tLPTS/HATS NPs were endowed with tumor recognition, internalization, penetration, and anti-invasion, and thus might be a promising anticancer drug delivery vehicle for targeted cancer therapy.     

In vivo multimodal magnetic particle imaging (MPI) with tailored magneto/optical contrast agents

Magnetic Particle Imaging (MPI) is a novel non-invasive biomedical imaging modality that uses safe magnetite nanoparticles as tracers. Controlled synthesis of iron oxide nanoparticles (NPs) with tuned size-dependent magnetic relaxation properties is critical for the development of MPI. Additional functionalization of these NPs for other imaging modalities (e.g. MRI and fluorescent imaging) would accelerate screening of the MPI tracers based on their in vitro and in vivo performance in pre-clinical trials. Here, we conjugated two different types of poly-ethylene-glycols (NH₂-PEG-NH₂ and NH₂-PEG-FMOC) to monodisperse carboxylated 19.7 nm NPs by amide bonding. Further, we labeled these NPs with Cy5.5 near infra-red fluorescent (NIRF) molecules. Bi-functional PEG (NH₂-PEG-NH₂) resulted in larger hydrodynamic size (∼98 nm vs. ∼43 nm) of the tracers, due to inter-particle crosslinking. Formation of such clusters impacted the multimodal imaging performance and pharmacokinetics of these tracers. We found that MPI signal intensity of the tracers in blood depends on their plasmatic clearance pharmacokinetics. Whole body mice MPI/MRI/NIRF, used to study the biodistribution of the injected NPs, showed primary distribution in liver and spleen. Biodistribution of tracers and their clearance pathway was further confirmed by MPI and NIRF signals from the excised organs where the Cy5.5 labeling enabled detailed anatomical mapping of the tracers.in tissue sections. These multimodal MPI tracers, combining the strengths of each imaging modality (e.g. resolution, tracer sensitivity and clinical use feasibility) pave the way for various in vitro and in vivo MPI applications.     

Intra-articular delivery of kartogenin-conjugated chitosan nano/microparticles for cartilage regeneration

We developed an intra-articular (IA) drug delivery system to treat osteoarthritis (OA) that consisted of kartogenin conjugated chitosan (CHI-KGN). Kartogenin, which promotes the selective differentiation of mesenchymal stem cells (MSCs) into chondrocytes, was conjugated with low-molecular-weight chitosan (LMWCS) and medium-molecular-weight chitosan (MMWCS) by covalent coupling of kartogenin to each chitosan using an ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) catalyst. Nanoparticles (NPs, 150 ± 39 nm) or microparticles (MPs, 1.8 ± 0.54 μm) were fabricated from kartogenin conjugated-LMWCS and –MMWCS, respectively, by an ionic gelation using tripolyphosphate (TPP). The in vitro release profiles of kartogenin from the particles showed sustained release for 7 weeks. When the effects of the CHI-KGN NPs or CHI-KGN MPs were evaluated on the in vitro chondrogenic differentiation of human bone marrow MSCs (hBMMSCs), the CHI-KGN NPs and CHI-KGN MPs induced higher expression of chondrogenic markers from cultured hBMMSCs than unconjugated kartogenin. In particular, hBMMSCs treated with CHI-KGN NPs exhibited more distinct chondrogenic properties in the long-term pellet cultures than those treated with CHI-KGN MPs. The in vivo therapeutic effects of CHI-KGN NPs or CHI-KGN MPs were investigated using a surgically-induced OA model in rats. The CHI-KGN MPs showed longer retention time in the knee joint than the CHI-KGN NPs after IA injection in OA rats. The rats treated with CHI-KGN NPs or CHI-KGN MPs by IA injection showed much less degenerative changes than untreated control or rats treated with unconjugated kartogenin. In conclusion, CHI-KGN NPs or CHI-KGN MPs can be useful polymer-drug conjugates as an IA drug delivery system to treat OA.     

A self-assembling nanoparticle for paclitaxel delivery in ovarian cancer

Paclitaxel (PTX) is one of the most effective chemotherapeutic drugs for the treatment of a variety of cancers. However, it is associated with serious side effects caused by PTX itself and the Cremophor EL emulsifier. In the present study, we report the development of a well-defined amphiphilic linear-dendritic copolymer (named as telodendrimer) composed of polyethylene glycol (PEG), cholic acid (CA, a facial amphiphilic molecule) and lysine, which can form drug-loaded core/shell micelles when mixed with hydrophobic drug, such as PTX, under aqueous condition. We have used PEG^5k-CA₈, a representive telodendrimer, to prepare paclitaxel-loaded nanoparticles (PTX-PEG^5k-CA₈ NPs) with high loading capacity (7.3 mg PTX/mL) and a size of 20–60 nm. This novel nanoformulation of PTX was found to exhibit similar in vitro cytotoxic activity against ovarian cancer cells as the free drug (Taxol^®) or paclitaxel/human serum albumin nanoaggregate (Abraxane^®). The maximum tolerated doses (MTDs) of PTX-PEG^5k-CA₈ NPs after single dose and five consecutive daily doses in mice were approximately 75 and 45 mg PTX/kg, respectively, which were 2.5-fold higher than those of Taxol^®. In both subcutaneous and orthotopic intraperitoneal murine models of ovarian cancer, PTX-PEG^5k-CA₈ NPs achieved superior toxicity profiles and anti-tumor effects compared to Taxol^® and Abraxane^® at equivalent PTX doses, which were attributed to their preferential tumor accumulation, and deep penetration into tumor tissue, as confirmed by near infrared fluorescence (NIRF) imaging.     

Tuning PEGylation of mixed micelles to overcome intracellular and systemic siRNA delivery barriers

A series of endosomolytic mixed micelles was synthesized from two diblock polymers, poly[ethylene glycol-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (PEG-b-pDPB) and poly[dimethylaminoethyl methacrylate-b-(dimethylaminoethyl methacrylate-co-propylacrylic acid-co-butyl methacrylate)] (pD-b-pDPB), and used to determine the impact of both surface PEG density and PEG molecular weight on overcoming both intracellular and systemic siRNA delivery barriers. As expected, the percent PEG composition and PEG molecular weight in the corona had an inverse relationship with mixed micelle zeta potential and rate of cellular internalization. Although mixed micelles were internalized more slowly, they generally produced similar gene silencing bioactivity (∼80% or greater) in MDA-MB-231 breast cancer cells as the micelles containing no PEG (100D/no PEG). The mechanistic explanation for the potent bioactivity of the promising 50 mol% PEG-b-DPB/50 mol% pD-b-pDPB (50D) mixed micelle formulation, despite its relatively low rate of cellular internalization, was further investigated as a function of PEG molecular weight (5 k, 10 k, or 20 k PEG). Results indicated that, although larger molecular weight PEG decreased cellular internalization, it improved cytoplasmic bioavailability due to increased intracellular unpackaging (quantitatively measured via FRET) and endosomal release. When delivered intravenously in vivo, 50D mixed micelles with a larger molecular weight PEG in the corona also demonstrated significantly improved blood circulation half-life (17.8 min for 20 k PEG micelles vs. 4.6 min for 5 kDa PEG micelles) and a 4-fold decrease in lung accumulation. These studies provide new mechanistic insights into the functional effects of mixed micelle-based approaches to nanocarrier surface PEGylation. Furthermore, the ideal mixed micelle formulation identified (50D/20 k PEG) demonstrated desirable intracellular and systemic pharmacokinetics and thus has strong potential for in vivo therapeutic use.     

One-step reduction and PEGylation of graphene oxide for photothermally controlled drug delivery

Here, we developed one-step green reduction and PEGylation of nanosized graphene oxide (NGO) to obtain NrGO/PEG as a photothermally controllable drug delivery system. NrGO/PEG was synthesized by bathing methoxypolyethylene glycol amine (mPEG-NH₂) and NGO at 90 °C for 24 h. The NrGO/PEG kept water stability for at least two months, and had ∼14-fold increment in near-infrared (NIR) absorbance and ∼2-fold increment in resveratrol (RV) loading over the unreduced NGO/PEG via π–π and hydrophobic interactions. Exposure of 4T1 cells to NrGO/PEG for 2 h showed 53.6% uptake ratio, and localization of NrGO/PEG in lysosomes instead of mitochondria. NIR irradiation (808 nm laser at 0.6 W/cm²) for 3 min potently enhanced RV release from NrGO/PEG-RV and the cytotoxicity of NrGO/PEG-RV against 4T1 cells, including decrease of cell viability, loss of mitochondrial membrane potential (ΔΨm) and cell apoptosis. Finally, NIR irradiation dramatically enhanced the efficacy of NrGO/PEG-RV in suppressing tumor growth in animal tumor models, further proving the remarkable synergistic action between photothermal effect of NrGO/PEG and RV loaded on NrGO/PEG.     

Long-circulating polymeric nanoparticles bearing a combinatorial coating of PEG and water-soluble chitosan

A major obstacle in the development of polymeric nanoparticles (NPs) as effective drug delivery vesicles is the rapid clearance from blood. In order to realize a significant prolongation in blood circulation, a combinatorial design, covalent attachment of polyethylene glycol (PEG) to polylactic acid (PLA) and physical adsorption of water-soluble chitosan (WSC) to particle surface, has been developed for surface modification of PLA NPs. Two types of WSC, cationic partially deacetylated chitin (PDC) and anionic N-carboxy propionyl chitosan sodium (CPCTS) were investigated. All the NPs formulated in the size range of 100–200 nm were prepared by a modified w/o/w technique and physicochemically characterized. In vitro phagocytosis by mouse peritoneal macrophage (MPM), in vivo blood clearance and biodistribution following intravenous administration in mice, of these NPs labeled with 6-coumarin, were evaluated. The presence of WSC, whether alone or with PEG, highly improved the surface hydrophilicity as well as suspension stability of NPs. Their surface charge was greatly affected by the WSC coating, being close to neutrality for PEG/PDC NPs and highly negative in the case of PEG/CPCTS NPs. In comparison to NPs treated with PEG or WSC alone, the synergistic action of PEG and WSC strongly inhibited the macrophage uptake and extended the circulation half-life (t_1/2) with concomitant reduced liver sequestration. Particularly, PEG/PDC NPs showed the most striking result with regard to their performance in vitro and in vivo. Calculated t_1/2 of PEG/PDC NPs and PEG/CPCTS NPs was 63.5 h and 7.1 h, respectively, much longer than that of control PEG/PVA NPs (1.1 h). More WSC materials need to be evaluated, but the present data suggest that, a combinatorial coating of PEG and PDC greatly prolongs the systemic circulation of NPs and represents a significant step in the development of long-circulating drug delivery carriers.     

A hydrogel-based tumor model for the evaluation of nanoparticle-based cancer therapeutics

Three-dimensional (3D) tissue-engineered tumor models have the potential to bridge the gap between monolayer cultures and patient-derived xenografts for the testing of nanoparticle (NP)-based cancer therapeutics. In this study, a hydrogel-derived prostate cancer (PCa) model was developed for the in vitro evaluation of doxorubicin (Dox)-loaded polymer NPs (Dox-NPs). The hydrogels were synthesized using chemically modified hyaluronic acid (HA) carrying acrylate groups (HA-AC) or reactive thiols (HA-SH). The crosslinked hydrogel networks exhibited an estimated pore size of 70–100 nm, similar to the spacing of the extracellular matrices (ECM) surrounding tumor tissues. LNCaP PCa cells entrapped in the HA matrices formed distinct tumor-like multicellular aggregates with an average diameter of 50 μm after 7 days of culture. Compared to cells grown on two-dimensional (2D) tissue culture plates, cells from the engineered tumoroids expressed significantly higher levels of multidrug resistance (MDR) proteins, including multidrug resistance protein 1 (MRP1) and lung resistance-related protein (LRP), both at the mRNA and the protein levels. Separately, Dox-NPs with an average diameter of 54 ± 1 nm were prepared from amphiphilic block copolymers based on poly(ethylene glycol) (PEG) and poly(ε-caprolactone) (PCL) bearing pendant cyclic ketals. Dox-NPs were able to diffuse through the hydrogel matrices, penetrate into the tumoroid and be internalized by LNCaP PCa cells through caveolae-mediated endocytosis and macropinocytosis pathways. Compared to 2D cultures, LNCaP PCa cells cultured as multicellular aggregates in HA hydrogel were more resistant to Dox and Dox-NPs treatments. Moreover, the NP-based Dox formulation could bypass the drug efflux function of MRP1, thereby partially reversing the resistance to free Dox in 3D cultures. Overall, the engineered tumor model has the potential to provide predictable results on the efficacy of NP-based cancer therapeutics.     

Development of high refractive ZnS/PVP/PDMAA hydrogel nanocomposites for artificial cornea implants

A series of high refractive index (RI) ZnS/PVP/PDMAA hydrogel nanocomposites containing ZnS nanoparticles (NPs) were successfully synthesized via a simple ultraviolet-light-initiated free radical co-polymerization method. The average diameter of the ZnS NPs is ∼3 nm and the NPs are well dispersed and stabilized in the PVP/PDMAA hydrogel matrix up to a high content of 60 wt.% in the hydrogel nanocomposites. The equilibrium water content of ZnS/PVP/PDMAA hydrogel nanocomposites varied from 82.0 to 66.8 wt.%, while the content of mercaptoethanol-capped ZnS NPs correspondingly varied from 30 to 60 wt.%. The resulting nanocomposites are clear and transparent and their RIs were measured to be as high as 1.58–1.70 and 1.38–1.46 in the dry and hydrated states, respectively, which can be tuned by varying the ZnS NPs content. In vitro cytotoxicity assays suggested that the introduction of ZnS NPs added little cytotoxicity to the PVP/PDMAA hydrogel and all the hydrogel nanocomposites exhibited minimal cytotoxicity towards common cells. The hydrogel nanocomposites implanted in rabbit eyes can be well tolerated over 3 weeks. Hence, the high RI ZnS/PVP/PDMAA hydrogel nanocomposites with adjustable RIs developed in this work might potentially be a candidate material for artificial corneal implants.     

Core-Shell type lipid/rPAA-Chol polymer hybrid nanoparticles for in vivo siRNA delivery

Our previous study had reported that cholesterol-grafted poly(amidoamine) (rPAA-Chol polymer) was able to self-assemble into cationic nanoparticles and act as a potential carrier for siRNA transfection. In this study, the core–shell type lipid/rPAA-Chol hybrid nanoparticles (PEG-LP/siRNA NPs and T7-LP/siRNA NPs) were developed for improving in vivo siRNA delivery by modifying the surface of rPAA-Chol/siRNA nanoplex core with a lipid shell, followed by post-insertion of polyethylene glycol phospholipid (DSPE-PEG) and/or peptide (HAIYPRH, named as T7) modified DSPE-PEG-T7. The integrative hybrid nanostructures of LP/siRNA NPs were evidenced by dynamic light scattering (DLS), confocal laser scanning microscope (CLSM), cryo-transmission electron microscope (Cryo-TEM) and surface plasmon resonance (SPR) assay. It was demonstrated that the T7 peptide modified LP/siRNA NPs (T7-LP/siRNA NPs) exhibited uniform and spherical structures with particle size of 99.39 ± 0.65 nm and surface potential of 42.53 ± 1.03 mV, and showed high cellular uptake efficiency and rapid endosomal/lysosomal escape ability in MCF-7 cells. Importantly, in vitro gene silencing experiment demonstrated that both of pegylated and targeted LP/siEGFR NPs exhibited significantly stronger downregulation of EGFR protein expression level in MCF-7 cells, compared to that of the physical mixture of siRNA lipoplexes and rPAA-Chol/siRNA nanoplexes. In vivo tumor therapy on nude mice bearing MCF-7 tumors further confirmed that the targeted T7-LP/siEGFR NPs exhibited the greatest inhibition on tumor growth via transferrin receptor-mediated targeting delivery, without any activation of immune responses and significant body weight loss following systemic administration. These findings indicated that the core-shell type T7-LP/siRNA nanoparticles would be promising siRNA delivery systems for in vivo tumor-targeted therapy.     

Co-delivery of chemotherapeutic drugs with vitamin E TPGS by porous PLGA nanoparticles for enhanced chemotherapy against multi-drug resistance

We report a strategy to make use of poly(lactic-co-glycolic acid) nanoparticle (PLGA NPs) for co-delivery of docetaxel (DTX) as a model anticancer drug together with vitamin E TPGS. The latter plays a dual role as a pore-forming agent in the nanoparticles that may result in smaller particle size, higher drug encapsulation efficiency and faster drug release, and also as a bioactive agent that could inhibit P-glycoprotein to overcome multi-drug resistance of the cancer cells, The DTX-loaded PLGA NPs of 0, 10, 20 and 40% TPGS were prepared by the nanoprecipitation method and then characterized for their size and size distribution, surface morphology, physical status and encapsulation efficiency of the drug in the NPs. All four NPs were found of size ranged 100–120 nm and EE ranged 85–95% at drug loading level around 10%. The in vitro evaluation showed that the 48 h IC₅₀ values of the free DTX and the DTX-loaded PLGA NPs of 0, 10, 20% TPGS were 2.619 and 0.474, 0.040, 0.009 μg/mL respectively, which means that the PLGA NPs formulation could be 5.57 fold effective than the free DTX and that the DTX-loaded PLGA NPs of 10 or 20% TPGS further be 11.85 and 52.7 fold effective than the DTX-loaded PLGA NPs of no TPGS (therefore, 66.0 and 284 fold effective than the free DTX). Xenograft tumor model and immunohistological staining analysis further confirmed the advantages of the strategy of co-delivery of anticancer drugs with TPGS by PLGA NPs.     

Inhibition of MDR1 gene expression and enhancing cellular uptake for effective colon cancer treatment using dual-surface-functionalized nanoparticles

Nanomedicine options for colon cancer therapy have been limited by the lack of suitable carriers capable of delivering sufficient drug into tumors to cause lethal toxicity. To circumvent this limitation, we fabricated a camptothecin (CPT)-loaded poly(lactic-co-glycolic acid) nanoparticle (NP) with dual-surface functionalization—Pluronic F127 and chitosan—for inhibiting multi-drug resistant gene 1 (MDR1) expression and enhancing tumor uptake. The resultant spherical NPs-P/C had a desirable particle size (∼268 nm), slightly positive zeta-potential, and the ability to efficiently down-regulate the expression of MDR1. In vitro cytotoxicity tests revealed that the 24 and 48 h IC₅₀ values of NPs-P/C1 were 2.03 and 0.67 μm, respectively, which were much lower than those for free CPT and other NPs. Interestingly, NPs-P/C1 showed the highest cellular uptake efficiency (approximately 85.5%) among the different drug formulations. Most importantly, treatment of colon tumor-bearing mice with various drug formulations confirmed that the introduction of Pluronic F127 and chitosan to the NP surface significantly enhanced the therapeutic efficacy of CPT, induced tumor cell apoptosis, and reduced systemic toxicity. Collectively, these findings suggest that our one-step-fabricated, dual-surface-functionalized NPs may hold promise as a readily scalable and effective drug carrier with clinical potential in colon cancer therapy.     

Enhanced blood–brain barrier penetration and glioma therapy mediated by a new peptide modified gene delivery system

Successful glioma gene therapy lays on two important factors, the therapeutic genes and efficient delivery vehicles to cross the blood–brain barrier (BBB) and reach gliomas. In this work, a new gene vector was constructed based on dendrigraft poly-l-lysines (DGL) and polyethyleneglycol (PEG), conjugated with a cell-penetrating peptide, the nucleolar translocation signal (NoLS) sequence of the LIM Kinase 2 (LIMK2) protein (LIMK2 NoLS peptide, LNP), yielding DGL-PEG-LNP. Plasmid DNA encoding inhibitor of growth 4 (ING4) was applied as the therapeutic gene. DGL-PEG-LNP/DNA nanoparticles (NPs) were monodispersed, with a mean diameter of 90.6 ± 8.9 nm. The conjugation of LNP significantly enhanced the BBB-crossing efficiency, cellular uptake and gene expression within tumor cells. Mechanism studies suggested the involvement of energy, caveolae-mediated endocytosis and macropinocytosis in cellular uptake of LNP-modified NPs. MTT results showed that no apparent cytotoxicity was observed when cells were treated with synthesized vectors. Furthermore, LNP-modified NPs mediated strongest and most intensive apoptosis on the tumor site, and the longest median survival time of glioma-bearing mice. All the results demonstrated that LNP is a kind of efficient CPPs especially for BBB-crossing application, and DGL-PEG-LNP/DNA is a potential non-viral platform for glioma gene therapy via intravenous administration.     

The penetration of fresh undiluted sputum expectorated by cystic fibrosis patients by non-adhesive polymer nanoparticles

Highly viscoelastic and adhesive sputum has precluded efficient nanoparticle-based drug and gene delivery to the lungs of patients with cystic fibrosis (CF). We sought to determine whether nanoparticles coated with non-mucoadhesive polymers could penetrate CF sputum, and to use these “muco-inert particles” (MIPs) as non-destructive nanoprobes to characterize the sputum microstructure. Particles as large as 200 nm in diameter that were densely coated with low MW polyethylene glycol (PEG) moved through undiluted CF sputum with average speeds up to 90-fold faster than similarly-sized uncoated particles. On the other hand, the transport of both coated and uncoated 500 nm particles was strongly hindered. The local viscosity of sputum, encountered by the fastest 10% of 200 nm MIPs, was only 5-fold higher than that of water, whereas the bulk viscosity was 10,000-fold higher at low shear rates. Using measured transport rates of various sized MIPs combined with an obstruction-scaling model, we determined that the average 3D mesh spacing of CF sputum is ～140 ± 50 nm (range: 60–300 nm). Taken together, these results demonstrate that nanoparticles up to 200 nm in diameter that do not adhere to CF sputum can move rapidly through this critical barrier by accessing pores that are filled with a low viscosity fluid. The results also offer hope that desperately needed sputum-penetrating drug- and gene-carrier nanoparticles can be developed for CF.     

Roles of ligand and TPGS of micelles in regulating internalization,penetration and accumulation against sensitive or resistant tumor and therapy for multidrug resistant tumors

There are several obstacles in the process of successful treatment of malignant tumors, including toxicity to normal cells, inefficiency of drug permeation and accumulation into the deep tissue of solid tumor, and multidrug resistance (MDR). In this work, we prepared docetaxel (DTX)-loaded hybrid micelles with DSPE–PEG and TPGS (TPGS/DTX-M), where TPGS serves as an effective P-gp inhibitor for overcoming MDR, and active targeting hybrid micelles (FA@TPGS/DTX-M) with targeting ligand of folate on the hybrid micelles surface offering active targeting to folate receptor-overexpressed tumor cells. A systematic comparative evaluation of these micelles on cellular internalization, sub-cellular distribution, antiproliferation, mitochondrial membrane potential, cell apoptosis and cell cycle, permeation and inhibition on 3-dimensional multicellular tumor spheroids, as well as antitumor efficacy and safety assay in vivo were well performed between sensitive KB tumors and resistant KBv tumors, and among P-gp substrate or not. We found that the roles of folate and TPGS varied due to the sensitivity of tumors and the loaded molecules in the micelles. Folate and folate receptor-mediated endocytosis played a leading role in internalization, permeation and accumulation for sensitive tumors and non-substrates of P-gp. On the contrary, TPGS played the predominant role which dramatically decreased the efflux of drugs both when the tumor is resistant and for P-gp substrate. These findings are very meaningful for guiding the design of carrier delivery system to treat tumors. The antitumor efficacy in xenograft nude mice model and safety assay showed that the TPGS/DTX-M and FA@TPGS/DTX-M significantly exhibited higher antitumor activity against resistant KBv tumors than the marketed formulation and normal micelles owing to the small size (approximately 20 nm), hydrophilic PEGylation, TPGS inhibition of P-gp function, and folate receptor-modified endocytosis, permeation and accumulation in solid tumor, as well as synergistic effects of DTX-induced cell division inhibition, growth restraint and TPGS-triggered mitochondrial apoptosis in tumor cells. In conclusion, folate-modified TPGS hybrid micelles provide a synergistic strategy for effective delivery of DTX into KBv cells and overcoming MDR.     

Doxorubicin-loaded glycyrrhetinic acid-modified alginate nanoparticles for liver tumor chemotherapy

Doxorubicin (DOX)-loaded glycyrrhetinic acid (GA)-modified alginate (ALG) nanoparticles (DOX/GA-ALG NPs) were prepared for targeting therapy of liver cancer. This study focused on the biodistribution of DOX/GA-ALG NPs in Kunming mice as well as their antitumor activity against liver tumors in situ and side effects. The biodistribution data showed that the concentration of DOX in the liver reached 67.8 ± 4.9 μg/g after intravenous administration of DOX/GA-ALG NPs, which was 2.8-fold and 4.7-fold higher compared to non-GA-modified nanoparticles (DOX/CHO-ALG NPs) and DOX·HCl, respectively. The concentration of DOX in the heart of mice treated with DOX/GA-ALG NPs at any sampling time was relatively lower than that of mice treated with DOX·HCl. The liver tumor growth inhibition rate (IR) in situ was about 52.6% and the mortality was 33% in DOX·HCl group. In contrast, the IR was 76.6% and no mice died in the DOX/GA-ALG NPs group. Histological examination showed tumor necrosis in both experimental groups. Most importantly, the heart cells and the liver cells surrounding the tumor were not affected by administration of DOX/GA-ALG NPs, whereas myocardial necrosis and apparent liver cell swelling were observed after DOX·HCl administration.     

Size effect of amphiphilic poly(γ-glutamic acid) nanoparticles on cellular uptake and maturation of dendritic cells in vivo

We prepared size-regulated nanoparticles (NPs) composed of amphiphilic poly(γ-glutamic acid) (γ-PGA). In this study, 40, 100 and 200 nm γ-PGA-graft-l-phenylalanine ethylester (γ-PGA-Phe) NPs were employed. The size of NPs significantly influenced the uptake and activation behaviors of antigen-presenting cells (APCs). When 40 nm γ-PGA-Phe NPs were applied to these cells in vitro, they were highly activated compared with 100 and 200 nm NPs, while cellular uptake was size dependent. The size of the γ-PGA-Phe NPs also significantly affected their migration to the lymph nodes and uptake behavior of NPs by dendritic cells (DCs) in vivo. The 40 nm γ-PGA-Phe NPs migrated more rapidly to the lymph nodes and were taken up by a greater number of DCs compared with 100 and 200 nm NPs. On the other hand, when the amount of γ-PGA-Phe NPs taken up per DC was evaluated, it was higher for 100 and 200 nm NPs than for 40 nm NPs, which suggests that the larger γ-PGA-Phe NPs can deliver a large amount of antigen to a single DC compared with smaller NPs. Furthermore, when examined the maturation of DCs in lymph nodes, 40 nm γ-PGA-Phe NPs efficiently stimulated DCs. These results suggest that the activation, uptake behavior by APCs, migration to lymph nodes, and DC maturation can be controlled by the size of γ-PGA-Phe NPs.     

Cationic core–shell nanoparticles with carmustine contained within O-benzylguanine shell for glioma therapy

The application of carmustine (BCNU) for glioma treatment is limited due to its poor selectivity for tumor and tumor resistance caused by O⁶-methylguanine-DNA-methyl transferase (MGMT). To improve the efficacy of BCNU, we constructed chitosan surface-modified poly (lactide-co-glycolides) nanoparticles (PLGA/CS NPs) for targeting glioma, loading BCNU along with O⁶-benzylguanine (BG), which could directly deplete MGMT. With core–shell structure, PLGA/CS NPs in the diameter around 177 nm showed positive zeta potential. In vitro plasma stability of BCNU in NPs was improved compared with free BCNU. The cellular uptake of NPs increased with surface modification of CS and decreasing particle size. The cytotoxicity of BCNU against glioblastoma cells was enhanced after being encapsulated into NPs; furthermore, with the co-encapsulation of BCNU and BG into NPs, BCNU + BG PLGA/CS NPs showed the strongest inhibiting ability. Compared to free drugs, PLGA/CS NPs could prolong circulation time and enhance accumulation in tumor and brain. Among all treatment groups, F98 glioma-bearing rats treated with BCNU + BG PLGA/CS NPs showed the longest survival time and the smallest tumor size. The studies suggested that the co-encapsulation of BCNU and BG into PLGA/CS NPs could remarkably enhance the efficacy of BCNU, accompanied with greater convenience for therapy.