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Introduction - Nanoparticles are increasingly used as drug carriers for oral administration. The delivery of drug molecules is largely dependent on the interaction of nanocarriers and gastrointestinal (GI) mucus, a critical barrier that regulates drug absorption. It is therefore important to understand the effects of physical and chemical properties of nanocarriers on the interaction with GI mucus. Unfortunately, most of the nanoparticles are unable to be prepared with satisfactory structural monodispersity to comprehensively investigate the interaction. With controlled size, shape, and surface chemistry, copolymers are ideal candidates for such purpose.
Materials and methods - We synthesized a series of diblock copolymers via the atom transfer radical polymerization method and investigated the GI mucus permeability in vitro and in vivo.
Results - Our results indicated that uncharged and hydrophobic copolymers exhibited enhanced GI absorption.
Conclusion - These results provide insights into developing optimal nanocarriers for oral administration.
Nanogels with a biomolecular coating (biocoating) were shown to be capable of triggered delivery of anticancer drug Doxorubicin. The biocoating was formed utilizing binding between glycogen and the tetra-functional lectin Concanavalin A, which can be triggered to disassemble (and release) upon exposure to glucose and changes in solution pH. We also show the nanogel's thermoresponsivity can be used to accelerate Doxorubicin release. Moreover, we showed that transferrin immobilized on the nanogel surface could accelerate nanogel uptake by cancer cells. In these experiments, we showed that Doxorubicin was able to be released to the nucleus of human liver cancer cell line (HepG2) within 3 h. Doxorubicin-loaded nanogels exhibit a strong growth inhibition ability toward HepG2. This investigation showcases how nanogel design and chemistry can be tuned to achieve useful biomedical applications.
Although siRNA-based nanomedicines hold promise for cancer treatment, conventional siRNA-polymer complex (polyplex) nanocarrier systems have poor pharmacokinetics following intravenous delivery, hindering tumor accumulation. Here, we determined the impact of surface chemistry on the in vivo pharmacokinetics and tumor delivery of siRNA polyplexes. A library of diblock polymers was synthesized, all containing the same pH-responsive, endosomolytic polyplex core-forming block but different corona blocks: 5 kDa (benchmark) and 20 kDa linear polyethylene glycol (PEG), 10 kDa and 20 kDa brush-like poly(oligo ethylene glycol), and 10 kDa and 20 kDa zwitterionic phosphorylcholine-based polymers (PMPC). In vitro, it was found that 20 kDa PEG and 20 kDa PMPC had the highest stability in the presence of salt or heparin and were the most effective at blocking protein adsorption. Following intravenous delivery, 20 kDa PEG and PMPC coronas both extended circulation half-lives 5-fold compared to 5 kDa PEG. However, in mouse orthotopic xenograft tumors, zwitterionic PMPC-based polyplexes showed highest in vivo luciferase silencing (>75% knockdown for 10 days with single IV 1 mg/kg dose) and 3-fold higher average tumor cell uptake than 5 kDa PEG polyplexes (20 kDa PEG polyplexes were only 2-fold higher than 5 kDa PEG). These results show that high molecular weight zwitterionic polyplex coronas significantly enhance siRNA polyplex pharmacokinetics without sacrificing polyplex uptake and bioactivity within tumors when compared to traditional PEG architectures.
Formation of stable, long-circulating siRNA polyplexes is a significant challenge in translation of intravenously-delivered, polymeric RNAi cancer therapies. Here, we report that siRNA hydrophobization through conjugation to palmitic acid (siPA) improves stability, in vivo pharmacokinetics, and tumor gene silencing of PEGylated nanopolyplexes (siPA-NPs) with balanced cationic and hydrophobic content in the core relative to the analogous polyplexes formed with unmodified siRNA, si-NPs. Hydrophobized siPA loaded into the NPs at a lower charge ratio (N(+):P(-)) relative to unmodified siRNA, and siPA-NPs had superior resistance to siRNA cargo unpackaging in comparison to si-NPs upon exposure to the competing polyanion heparin and serum. In vitro, siPA-NPs increased uptake in MDA-MB-231 breast cancer cells (100% positive cells vs. 60% positive cells) but exhibited equivalent silencing of the model gene luciferase relative to si-NPs. In vivo in a murine model, the circulation half-life of intravenously-injected siPA-NPs was double that of si-NPs, resulting in a >2-fold increase in siRNA biodistribution to orthotopic MDA-MB-231 mammary tumors. The increased circulation half-life of siPA-NPs was dependent upon the hydrophobic interactions of the siRNA and the NP core component and not just siRNA hydrophobization, as siPA did not contribute to improved circulation time relative to unmodified siRNA when delivered using polyplexes with a fully cationic core. Intravenous delivery of siPA-NPs also achieved significant silencing of the model gene luciferase in vivo (∼40% at 24 h after one treatment and ∼60% at 48 h after two treatments) in the murine MDA-MB-231 tumor model, while si-NPs only produced a significant silencing effect after two treatments. These data suggest that stabilization of PEGylated siRNA polyplexes through a combination of hydrophobic and electrostatic interactions between siRNA cargo and the polymeric carrier improves in vivo pharmacokinetics and tumor gene silencing relative to conventional formulations that are stabilized solely by electrostatic interactions.
Copyright © 2016 Elsevier Ltd. All rights reserved.
We report the synthesis and encapsulation of polyester nanosponge particles (NPs) co-loaded with tamoxifen (TAM) and quercetin (QT) to investigate the loading, release and in vitro metabolism of a dual drug formulation. The NPs are made in two variations, 4% and 8% crosslinking densities, to evaluate the effects on metabolism and release kinetics. The NP-4% formulation with a particle size of 89.3 ± 14.8 nm was found to have loading percentages of 6.91 ± 0.13% TAM and 7.72 ± 0.15% QT after targeting 10% (w/w) each. The NP-8% formulation with a particle size of 91.5 ± 9.8 nm was found to have loading percentages of 7.26 ± 0.10% TAM and 7.80 ± 0.12% QT. The stability of the formulation was established in simulated gastrointestinal fluids, and the metabolism of TAM was shown to be reduced 2-fold and 3-fold for NP-4%s and NP-8%s, respectively, while QT metabolism was reduced 3 and 4-fold. The implications for improved bioavailability of the NP formulations were supported by cytotoxicity results that showed a similar efficacy to free dual drug formulations and even enhanced anti-cancer effects in the recovery condition. This work demonstrates the suitability of the nanosponges not only as a dual release drug delivery system but also enabling a regulated metabolism through the capacity of a nanonetwork. The variation in crosslinking enables a dual release with tailored release kinetics and suggests improved bioavailability aided by a reduced metabolism.
Copyright © 2015 Elsevier B.V. All rights reserved.
A versatile and scalable method for fabricating shape-engineered nano- and micrometer scale particles from mesoporous silicon (PSi) thin films is presented. This approach, based on the direct imprinting of porous substrates (DIPS) technique, facilitates the generation of particles with arbitrary shape, ranging in minimum dimension from approximately 100 nm to several micrometers, by carrying out high-pressure (>200 MPa) direct imprintation, followed by electrochemical etching of a sub-surface perforation layer and ultrasonication. PSi particles (PSPs) with a variety of geometries have been produced in quantities sufficient for biomedical applications (≫10 μg). Because the stamps can be reused over 150 times, this process is substantially more economical and efficient than the use of electron beam lithography and reactive ion etching for the fabrication of nanometer-scale PSPs directly. The versatility of this fabrication method is demonstrated by loading the DIPS-imprinted PSPs with a therapeutic peptide nucleic acid drug molecule, and by vapor deposition of an Au coating to facilitate the use of PSPs as a photothermal contrast agent.
In regions of the circulation where vessels are straight and unbranched, blood flow is laminar and unidirectional. In contrast, at sites of curvature, branch points, and regions distal to stenoses, blood flow becomes disturbed. Atherosclerosis preferentially develops in these regions of disturbed blood flow. Current therapies for atherosclerosis are systemic and may not sufficiently target these atheroprone regions. In this study, we sought to leverage the alterations on the luminal surface of endothelial cells caused by this atheroprone flow for nanocarrier targeting. In vivo phage display was used to discover unique peptides that selectively bind to atheroprone regions in the mouse partial carotid artery ligation model. The peptide GSPREYTSYMPH (PREY) was found to bind 4.5-fold more avidly to the region of disturbed flow and was used to form targeted liposomes. When administered intravenously, PREY-targeted liposomes preferentially accumulated in endothelial cells in the partially occluded carotid artery and other areas of disturbed flow. Proteomic analysis and immunoblotting indicated that fibronectin and Filamin-A were preferentially bound by PREY nanocarriers in vessels with disturbed flow. In additional experiments, PREY nanocarriers were used therapeutically to deliver the nitric oxide synthase cofactor tetrahydrobiopterin (BH4), which we have previously shown to be deficient in regions of disturbed flow. This intervention increased vascular BH4 and reduced vascular superoxide in the partially ligated artery in wild-type mice and reduced plaque burden in the partially ligated left carotid artery of fat fed atheroprone mice (ApoE(-/-)). Targeting atheroprone sites of the circulation with functionalized nanocarriers provides a promising approach for prevention of early atherosclerotic lesion formation.
Tumor associated macrophages (TAMs) can modify the tumor microenvironment to create a pro-tumor niche. Manipulation of the TAM phenotype is a novel, potential therapeutic approach to engage anti-cancer immunity. siRNA is a molecular tool for knockdown of specific mRNAs that is tunable in both strength and duration. The use of siRNA to reprogram TAMs to adopt an immunogenic, anti-tumor phenotype is an attractive alternative to ablation of this cell population. One current difficulty with this approach is that TAMs are difficult to specifically target and transfect. We report here successful utilization of novel mannosylated polymer nanoparticles (MnNP) that are capable of escaping the endosomal compartment to deliver siRNA to TAMs in vitro and in vivo. Transfection with MnNP-siRNA complexes did not significantly decrease TAM cell membrane integrity in culture, nor did it create adverse kidney or liver function in mice, even at repeated doses of 5 mg kg(-1). Furthermore, MnNP effectively delivers labeled nucleotides to TAMs in mice with primary mammary tumors. We also confirmed TAM targeting in the solid tumors disseminated throughout the peritoneum of ovarian tumor bearing mice following injection of fluorescently labeled MnNP-nucleotide complexes into the peritoneum. Finally, we show enhanced uptake of MnNP in lung metastasis associated macrophages compared to untargeted particles when using an intubation delivery method. In summary, we have shown that MnNP specifically and effectively deliver siRNA to TAMs in vivo.
A combination of anionic and RAFT polymerization was used to synthesize an ABC triblock polymer poly[(propylenesulfide)-block-(N,N-dimethylacrylamide)-block-(N-isopropylacrylamide)] (PPS-b-PDMA-b-PNIPAAM) that forms physically cross-linked hydrogels when transitioned from ambient to physiologic temperature and that incorporates mechanisms for reactive oxygen species (ROS) triggered degradation and drug release. At ambient temperature (25 °C), PPS-b-PDMA-b-PNIPAAM assembled into 66 ± 32 nm micelles comprising a hydrophobic PPS core and PNIPAAM on the outer corona. Upon heating to physiologic temperature (37 °C), which exceeds the lower critical solution temperature (LCST) of PNIPAAM, micelle solutions (at ≥2.5 wt %) sharply transitioned into stable, hydrated gels. Temperature-dependent rheology indicated that the equilibrium storage moduli (G') of hydrogels at 2.5, 5.0, and 7.5 wt % were 20, 380, and 850 Pa, respectively. The PPS-b-PDMA-b-PNIPAAM micelles were preloaded with the model drug Nile red, and the resulting hydrogels demonstrated ROS-dependent drug release. Likewise, exposure to the peroxynitrite generator SIN-1 degraded the mechanical properties of the hydrogels. The hydrogels were cytocompatible in vitro and were demonstrated to have utility for cell encapsulation and delivery. These hydrogels also possessed inherent cell-protective properties and reduced ROS-mediated cellular death in vitro. Subcutaneously injected PPS-b-PDMA-b-PNIPAAM polymer solutions formed stable hydrogels that sustained local release of the model drug Nile red for 14 days in vivo. These collective data demonstrate the potential use of PPS-b-PDMA-b-PNIPAAM as an injectable, cyto-protective hydrogel that overcomes conventional PNIPAAM hydrogel limitations such as syneresis, lack of degradability, and lack of inherent drug loading and environmentally responsive release mechanisms.
Peripheral artery disease (PAD) is characterized by vessel occlusion and ischemia in the limbs. Treatment for PAD with surgical interventions has been showing limited success. Moreover, recent clinical trials with treatment of angiogenic growth factors proved ineffective as increased angiogenesis triggered severe inflammation in a proportionally coupled fashion. Hence, the overarching goal of this research was to address this issue by developing a biomaterial system that enables controlled, dual delivery of pro-angiogenic C16 and anti-inflammatory Ac-SDKP peptides in a minimally-invasive way. To achieve the goal, a peptide-loaded injectable microgel system was developed and tested in a mouse model of PAD. When delivered through multiple, low volume injections, the combination of C16 and Ac-SDKP peptides promoted angiogenesis, muscle regeneration, and perfusion recovery, while minimizing detrimental inflammation. Additionally, this peptide combination regulated inflammatory TNF-α pathways independently of MMP-9 mediated pathways of angiogenesis in vitro, suggesting a potential mechanism by which angiogenic and inflammatory responses can be uncoupled in the context of PAD. This study demonstrates a translatable potential of the dual peptide-loaded injectable microgel system for PAD treatment.
Copyright © 2014 Elsevier Ltd. All rights reserved.