Overcoming endosomal barrier by amphotericin B-loaded dual pH-responsive PDMA-b-PDPA micelleplexes for siRNA delivery. ACS Nano 5:9246-9255

Department of Pharmacology, Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390, USA.
ACS Nano (Impact Factor: 12.88). 11/2011; 5(11):9246-55. DOI: 10.1021/nn203503h
Source: PubMed

ABSTRACT The endosomal barrier is a major bottleneck for the effective intracellular delivery of siRNA by nonviral nanocarriers. Here, we report a novel amphotericin B (AmB)-loaded, dual pH-responsive micelleplex platform for siRNA delivery. Micelles were self-assembled from poly(2-(dimethylamino)ethyl methacrylate)-block-poly(2-(diisopropylamino)ethyl methacrylate) (PDMA-b-PDPA) diblock copolymers. At pH 7.4, AmB was loaded into the hydrophobic PDPA core, and siRNA was complexed with a positively charged PDMA shell to form the micelleplexes. After cellular uptake, the PDMA-b-PDPA/siRNA micelleplexes dissociated in early endosomes to release AmB. Live cell imaging studies demonstrated that released AmB significantly increased the ability of siRNA to overcome the endosomal barrier. Transfection studies showed that AmB-loaded micelleplexes resulted in significant increase in luciferase (Luc) knockdown efficiency over the AmB-free control. The enhanced Luc knockdown efficiency was abolished by bafilomycin A1, a vacuolar ATPase inhibitor that inhibits the acidification of the endocytic organelles. These data support the central hypothesis that membrane poration by AmB and increased endosomal swelling and membrane tension by a "proton sponge" polymer provided a synergistic strategy to disrupt endosomes for improved intracellular delivery of siRNA.

Download full-text


Available from: Yiguang Wang, Sep 29, 2015
60 Reads
  • Source
    • "It was envisioned that retaining the PEG component of these polymers would be important not only for micelle assembly but also to retard internalization of the NPs following surface conjugation and to minimize the perturbation of the surface properties of the cells – as coupling just PDPAEMA NPs to the surface would introduce islands of cationic density. While PDPAEMA has been used as a component of pH-responsive NPs for delivering siRNA [41], it was unknown if the biotinylated PEG- PDPAEMA would be effective in releasing a small molecule in a pHdependent fashion. To assess this, biotinylated NPs were loaded with nile red, a fluorescently active model small (340 Da), hydrophobic drug (Fig. 2a). "
    [Show abstract] [Hide abstract]
    ABSTRACT: Intratumoral drug delivery is an inherently appealing approach for concentrating toxic chemotherapies at the site of action. This mode of administration is currently used in a number of clinical treatments such as neoadjuvant, adjuvant, and even standalone therapies when radiation and surgery are not possible. However, even when injected locally, it is difficult to achieve efficient distribution of chemotherapeutics throughout the tumor. This is primarily attributed to the high interstitial pressure which results in gradients that drive fluid away from the tumor center. The stiff extracellular matrix also limits drug penetration throughout the tumor. We have previously shown that neural stem cells can penetrate tumor interstitium, actively migrating even to hypoxic tumor cores. When used to deliver therapeutics, these migratory neural stem cells result in dramatically enhanced tumor coverage relative to conventional delivery approaches. We recently showed that neural stem cells maintain their tumor tropic properties when surface-conjugated to nanoparticles. Here we demonstrate that this hybrid delivery system can be used to improve the efficacy of docetaxel-loaded nanoparticles when administered intratumorally. This was achieved by conjugating drug-loaded nanoparticles to the surface of neural stem cells using a bond that allows the stem cells to efficiently distribute nanoparticles throughout the tumor before releasing the drug for uptake by tumor cells. The modular nature of this system suggests that it could be used to improve the efficacy of many chemotherapy drugs after intratumoral administration.
    Journal of Controlled Release 06/2014; 191. DOI:10.1016/j.jconrel.2014.06.015 · 7.71 Impact Factor
  • Source
    • "There are examples of agents that have been demonstrated specifically to enhance endosomal release of co-delivery micelleplexes. Yu and colleagues created poly(2-(dimethylamino)ethyl methacrylate)-block- poly(2-(diisopropylamino)ethyl methacrylate) (PDMA-b-PDPA) copolymer micelles complexed with siRNA and loaded with amphotericin B (Yu et al., 2011). Amphotericin B is an antifungal drug which creates pores in cell membranes, and its release was shown to increase the delivery of siRNA by overcoming the endosomal barrier. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Tumor cells exhibit drug resistant phenotypes that decrease the efficacy of chemotherapeutic treatments. The drug resistance has a genetic basis that is caused by an abnormal gene expression. There are several types of drug resistance: efflux pumps reducing the cellular concentration of the drug, alterations in membrane lipids that reduce cellular uptake, increased or altered drug targets, metabolic alteration of the drug, inhibition of apoptosis, repair of the damaged DNA, and alteration of the cell cycle checkpoints (Gottesman et al. , 2002, Holohan et al. , 2013). siRNA is used to silence the drug resistant phenotype and prevent this drug resistance response. Of the listed types of drug resistance, pump-type resistance (e.g., high expression of ATP-binding cassette transporter proteins such as P-glycoproteins (Pgp; also known as multi-drug resistance protein 1 or MDR1)) and apoptosis inhibition (e.g., expression of anti-apoptotic proteins such as Bcl-2) are the most frequently targeted for gene silencing. The co-delivery of siRNA and chemotherapeutic drugs has a synergistic effect, but many of the current projects do not control the drug release from the nanocarrier. This means the drug payload is released before the drug resistance proteins have degraded and the drug resistance phenotype has been silenced. Current research focuses on cross-linking the carrier's polymers to prevent premature drug release, but these carriers still rely on environmental cues to release the drug payload, and the drug may be released too early. In this review, we studied the release kinetics of siRNA and chemotherapeutic drugs from a broad range of carriers. We also give examples of carriers used to co-deliver siRNA and drugs to drug-resistant tumor cells, and we examine how modifications to the carrier affect the delivery. Lastly, we give our recommendations for the future directions of the co-delivery of siRNA and chemotherapeutic drug treatments.
    Biotechnology Advances 06/2014; 32(5). DOI:10.1016/j.biotechadv.2014.05.006 · 9.02 Impact Factor
  • Source
    • "Importantly, the net cationic charge displayed by the PNP can be modulated simply by changing the molar ratios of P1:P2. This mixed micelle approach negates the need for extensive and time-consuming resynthesis of PNPs as, unlike triblock copolymers (ABC or ABA) [23] [24] [25], and single diblock copolymers (AB) [26] [27], the two independent amphiphilic diblock copolymers (AB and CB) open the possibility of constructing rapidly numerous mixed micelle compositions. Eleven PNPs were prepared (Table 1), in which the molar ratio of P1: P2 was changed from 100:0 to 0:100 in 10% intervals to afford PNP0– PNP100, where the number indicates the percentage of cationic polymer P1 chain within the mixed micelle. "
    [Show abstract] [Hide abstract]
    ABSTRACT: Short interfering ribonucleic acids (siRNAs) offer a highly specific and selective form of therapy for diseases with a genetic component; however the poor pharmacokinetic properties of the molecule have impeded its development into a therapeutic for use in vivo. Several different approaches have been taken to develop a successful siRNA delivery system but these systems lack the flexibility for easy optimisation. Here, we propose a polymeric nanoparticle (PNP) system consisting of two amphiphilic diblock copolymers which allow for the rapid determination of structure-activity relationships involving gene knockdown and toxicity. The diblock copolymers self-assemble into monodisperse micelles of defined hydrodynamic diameters ranging from 30 to 100nm dependent on the copolymer ratio. A luciferase-based high throughput assay varying PNP composition, concentration and siRNA concentration allowed the rapid identification of efficient PNP formulations for adherent and suspension cell lines. Optimised PNPs efficiently knocked down a fusion oncogene in hard to transfect human leukaemic cells raising the possibility of targeting malignant cells in a cancer-specific fashion. This approach allows the optimum PNP formulation to be identified for different cell types and conditions.
    Journal of Controlled Release 10/2013; 172(3). DOI:10.1016/j.jconrel.2013.10.013 · 7.71 Impact Factor
Show more