Exploring the outer limits of polyplexes.

Histidine-containing polymers show promise in their transport of nucleic acids in vitro and in vivo. In addition to the pH-buffering histidine component, the polymer often contains a protonated component at physiological pH, such as lysine. These polyplexes usually accumulate in the tumor by enhanced permeability and retention, which has proved disappointing in clinical trials. We presently compare two histidine-lysine (HK) peptide polyplexes for their neuropilin-1-mediated transport of plasmids in vivo. While the polymerized HK (H2KC-48) polyplex was markedly better than the monomeric HK (H2K) polyplex in vitro, both HK polyplexes were effective in transfecting tumor xenografts over a wide range of peptide and plasmid ratios. Nevertheless, polyplexes of low peptide/DNA ratios gave higher tumor transfection and specificity than those of higher ratios. Surprisingly, there was minimal to no gel retardation of polyplexes made from these low ratios during electrophoresis. These results demonstrate that loosely packed HK polyplexes effectively transfected tumors in vivo.

Hyaluronic acid-functionalized lipoplexes and polyplexes as emerging nanocarriers for receptor-targeted cancer therapy.

Cancer is an intricate disease that develops as a response to a combination of hereditary and environmental risk factors, which then result in a variety of changes to the genome. The cluster of differentiation (CD44) is a type of transmembrane glycoprotein that serves as a potential biomarker for cancer stem cells (CSC) and viable targets for therapeutic intervention in the context of cancer therapy. Hyaluronic acid (HA) is a linear polysaccharide that exhibits a notable affinity for the CD44 receptor. This characteristic renders it a promising candidate for therapeutic interventions aimed at selectively targeting CD44-positive cancer cells. Treating cancer via non-viral vector-based gene delivery has changed the notion of curing illness through the incorporation of therapeutic genes into the organism. The objective of this review is to provide an overview of various hyaluronic acid-modified lipoplexes and polyplexes as potential drug delivery methods for specific forms of cancer by effectively targeting CD44.

Peptide-Based Nanoparticles for Systemic Extrahepatic Delivery of Therapeutic Nucleotides.

Peptide-based nanoparticles (PBN) for nucleotide complexation and targeting of extrahepatic diseases are gaining recognition as potent pharmaceutical vehicles for fine-tuned control of protein production (up- and/or down-regulation) and for gene delivery. Herein, we review the principles and mechanisms underpinning self-assembled formation of PBN, cellular uptake, endosomal release, and delivery to extrahepatic disease sites after systemic administration. Selected examples of PBN that have demonstrated recent proof of concept in disease models in vivo are summarized to offer the reader a comparative view of the field and the possibilities for clinical application.

CRISPR/Cas9 Mutagenesis through Introducing a Nanoparticle Complex Made of a Cationic Polymer and Nucleic Acids into Maize Protoplasts.

Presently, targeted gene mutagenesis attracts increasing attention both in plant research and crop improvement. In these approaches, successes are largely dependent on the efficiency of the delivery of gene editing components into plant cells. Here, we report the optimization of the cationic polymer poly(2-hydroxypropylene imine) (PHPI)-mediated delivery of plasmid DNAs, or single-stranded oligonucleotides labelled with Cyanine3 (Cy3) or 6-Carboxyfluorescein (6-FAM)-fluorescent dyes into maize protoplasts. Co-delivery of the GFP-expressing plasmid and the Cy3-conjugated oligonucleotides has resulted in the cytoplasmic and nuclear accumulation of the green fluorescent protein and a preferential nuclear localization of oligonucleotides. We show the application of nanoparticle complexes, i.e., "polyplexes" that comprise cationic polymers and nucleic acids, for CRISPR/Cas9 editing of maize cells. Knocking out the functional EGFP gene in transgenic maize protoplasts was achieved through the co-delivery of plasmids encoding components of the editing factors Cas9 (pFGC-pcoCas9) and gRNA (pZmU3-gRNA) after complexing with a cationic polymer (PHPI). Several edited microcalli were identified based on the lack of a GFP fluorescence signal. Multi-base and single-base deletions in the EGFP gene were confirmed using Sanger sequencing. The presented results support the use of the PHPI cationic polymer in plant protoplast-mediated genome editing approaches.

Polysaccharide-Polyplex Nanofilm Coatings Enhance Nanoneedle-Based Gene Delivery and Transfection Efficiency.

Non-viral vectors represent versatile and immunologically safer alternatives for nucleic acid delivery. Nanoneedles and high-aspect ratio nanostructures are unconventional but interesting delivery systems, in which delivery is mediated by surface interactions. Herein, nanoneedles are synergistically combined with polysaccharide-polyplex nanofilms and enhanced transfection efficiency is observed, compared to polyplexes in suspension. Different polyplex-polyelectrolyte nanofilm combinations are assessed and it is found that transfection efficiency is enhanced when using polysaccharide-based polyanions, rather than being only specific for hyaluronic acid, as suggested in earlier studies. Moreover, results show that enhanced transfection is not mediated by interactions with the CD44 receptor, previously hypothesized as a major mechanism mediating enhancement via hyaluronate. In cardiac tissue, nanoneedles are shown to increase the transfection efficiency of nanofilms compared to flat substrates; while in vitro, high transfection efficiencies are observed in nanostructures where cells present large interfacing areas with the substrate. The results of this study demonstrate that surface-mediated transfection using this system is efficient and safe, requiring amounts of nucleic acid with an order of magnitude lower than standard culture transfection. These findings expand the spectrum of possible polyelectrolyte combinations that can be used for the development of suitable non-viral vectors for exploration in further clinical trials.

Optimization of Lung Surfactant Coating of siRNA Polyplexes for Pulmonary Delivery.

PURPOSE: The aim of this study was to understand how coating with a pulmonary surfactant, namely Alveofact, affects the physicochemical parameters as well as in vitro behavior of polyethylenimine (PEI) polyplexes for pulmonary siRNA delivery. METHODS: Alveofact-coated polyplexes were prepared at different Alveofact:PEI coating ratios and analyzed in terms of size, PDI and zeta potential as well as morphology by transmission electron microscopy. The biological behavior was evaluated in a lung epithelial cell line regarding cell viability, cellular uptake via flow cytometry and gene downregulation by qRT-PCR. Furthermore, a 3D ALI culture model was established to test the mucus diffusion and cellular uptake by confocal microscopy as well as gene silencing activity by qRT-PCR. RESULTS: After optimizing the coating process by testing different Alveofact:PEI coating ratios, a formulation with suitable parameters for lung delivery was obtained. In lung epithelial cells, Alveofact-coated polyplexes were well tolerated and internalized. Furthermore, the coating improved the siRNA-mediated gene silencing efficiency. Alveofact-coated polyplexes were then tested on a 3D air-liquid interface (ALI) culture model that, by expressing tight junctions and secreting mucus, resembles important traits of the lung epithelium. Here, we identified the optimal Alveofact:PEI coating ratio to achieve diffusion through the mucus layer while retaining gene silencing activity. Interestingly, the latter underlined the importance of establishing appropriate in vitro models to achieve more consistent results that better predict the in vivo activity. CONCLUSION: The addition of a coating with pulmonary surfactant to polymeric cationic polyplexes represents a valuable formulation strategy to improve local delivery of siRNA to the lungs.

Random Copolymers of Lysine and Isoleucine for Efficient mRNA Delivery.

Messenger RNA (mRNA) is currently of great interest as a new category of therapeutic agent, which could be used for prevention or treatment of various diseases. For this mRNA requires effective delivery systems that will protect it from degradation, as well as allow cellular uptake and mRNA release. Random poly(lysine-co-isoleucine) polypeptides were synthesized and investigated as possible carriers for mRNA delivery. The polypeptides obtained under lysine:isoleucine monomer ratio equal to 80/20 were shown to give polyplexes with smaller size, positive ζ-potential and more than 90% encapsulation efficacy. The phase inversion method was proposed as best way for encapsulation of mRNA into polyplexes, which are based on obtained amphiphilic copolymers. These copolymers showed efficacy in protection of bound mRNA towards ribonuclease and lower toxicity as compared to lysine homopolymer. The poly(lysine-co-isoleucine) polypeptides showed greater than poly(ethyleneimine) efficacy as vectors for transfection of cells with green fluorescent protein and firefly luciferase encoding mRNAs. This allows us to consider obtained copolymers as promising candidates for mRNA delivery applications.

Role of small interfering RNA (siRNA) in targeting ocular neovascularization: A review.

Ocular neovascularization (NV) plays a central role in the pathogenesis of various ocular diseases including diabetic retinopathy, age-related macular degeneration, retinoblastoma, retinitis pigmentosa and may lead to loss of vision if not controlled in time. Several clinical trials elucidate the central role of vascular endothelial growth factor (VEGF) in the pathogenesis of the ocular neovascularization. The advent and extensive use of ocular anti-VEGF therapy heralded a new age in the treatment of retinal vascular and exudative diseases. RNA interference (RNAi) can be used to inhibit the in-vitro and in-vivo expression of specific genes and thus provides an extremely useful method for investigating gene activity with minimal toxicity. siRNA targeting VEGF overcomes many drawbacks associated with the conventional treatment available for the treatment of ocular neovascularization. However, delivery methods that protect the siRNA against degradation and are appropriate for long-term care will help increase the effectiveness of RNAi-based anti-VEGF ocular therapies. Several nanotechnology approaches have been explored by formulation scientists for delivery of siRNA to the eye; targeting particularly VEGF for the treatment of NV. This review mainly focuses on current updates in various pre-clinical and clinical siRNA strategies for targeting VEGF involved in the development of ocular neovascularization.

Fabrication of antigen-containing nanoparticles using microfluidics with Tesla structure.

The polyethyleneimine (PEI)-based antigen delivery system has been proved valuable for therapeutic vaccines. However, the previous bulk mixing method is not ideal to prepare PEI-base antigen- containing nanoparticles. The wide-size distribution and poor reproducibility limit its further application. In this research, we developed a microfluidic method to prepare nanopolyplexes on chips with Tesla structure to improve the fabrication. The structure and bioactivity of protein were not hampered by the shearing force in microfluidics. Comparison between physiochemical parameters suggested that the polyplexes prepared by Tesla chips were more uniform than those prepared by bulk mixing and non-Tesla chips. The reproducibility was improved obviously. The preparation was not influence much by the operating parameters such as flow rates, reagents concentration, and switching of inlets. The results indicated that it was a robust and reliable method. The data that were obtained from BMDC model demonstrated that the nanopolyplexes with optimal weight ratio had higher antigen cross-presentation efficiency than those in free antigen group. In conclusion, Tesla structured microfluidics offers a better method over bulk mixing in the preparation of PEI-based antigen-containing nanopolyplexes. It greatly expands the scope of our study and increases the potential of PEI-based antigen delivery system.

DNA Polyplexes of a Phosphorylcholine-Based Zwitterionic Polymer for Gene Delivery.

PURPOSE: We tested polyplexes of a diblock polymer containing a pH-responsive, endosomolytic core (dimethylaminoethyl methacrylate and butyl methacrylate; DB) and a zwitterionic Poly (methacryloyloxyethyl phosphorylcholine) (PMPC) corona for the delivery of plasmid DNA (pDNA) to glioblastoma cells. METHODS: We studied the physicochemical characteristics of the DNA polyplexes such as particle hydrodynamic diameter and surface potential. Cytocompatibility of free PMPC-DB polymer and pDNA polyplexes with U-87MG and U-138MG glioma cell lines were evaluated using the ATP assay. The transfection activity of luciferase pDNA polyplexes was measured using a standard luciferase assay. Anti-proliferative, apoptotic, and cell migration inhibitory activities of PMPC-DB/Interferon-beta (IFN-ß1) pDNA polyplexes were examined using ATP assay, flow cytometry, and wound closure assay, respectively. RESULTS: PMPC-DB copolymer condensed pDNA into nanosized polyplexes. DNA polyplexes showed particle diameters ranging from ca. 100-150 nm with narrow polydispersity indices and near electroneutral zeta potential values. PMPC-DB/Luciferase pDNA polyplexes were safe and showed an 18-fold increase in luciferase expression compared to the gold standard PEI polyplexes in U-87MG cells. PMPC-DB/IFN-ß1 polyplexes induced apoptosis, demonstrated anti-proliferative effects, and retarded cell migration in glioblastoma cells. CONCLUSION: The results described herein should guide the future optimization of PMPC-DB/DNA delivery systems for in vivo studies.

Cylindrical Zwitterionic Particles via Interpolyelectrolyte Complexation on Molecular Polymer Brushes.

The fabrication of macromolecular architectures with high aspect ratio and well-defined internal and external morphologies remains a challenge. The combination of template chemistry and self-assembly concepts to construct peculiar polymer architectures via a bottom-up approach is an emerging approach. In this study, a cylindrical template-namely a core-shell molecular polymer brush-and linear diblock copolymers (DBCP) associate to produce high aspect ratio polymer particles via interpolyelectrolyte complexation. Induced, morphological changes are studied using cryogenic transmission electron and atomic force microscopy, while the complexation is further followed by isothermal titration calorimetry and ξ-potential measurements. Depending on the nature of the complexing DBCP, distinct morphological differences can be achieved. While polymers with a non-ionic block lead to internal compartmentalization, polymers featuring zwitterionic domains lead to a wrapping of the brush template.

Bioreducible crosslinked cationic nanopolyplexes from clickable polyethylenimines enabling robust cancer gene therapy.

Bioreducible crosslinked polyplexes from branched polyethylenimine (BPEI, 10 kDa) were successfully constructed through DNA neutralization by disulfide-linked azidated BPEI (PAZ) and subsequent DNA condensation by azadibenzocyclooctyne-modified BPEI (PDB), following their self-crosslinking via azide-azadibenzocyclooctyne click chemistry. Click-crosslinked cationic polyplexes (c-polyplexes) revealed high extracellular colloidal stability against negative heparin and ions while intracellular bioreducible degradability for efficient gene unpacking. In vitro gene transfection in cancer cells indicated that the c-polyplexes produced markedly higher transfection efficiency than non-crosslinked counterparts in the serum. The c-polyplexes also had prolonged circulation kinetics, elevated gene accumulation level in SKOV-3 tumor xenografted in a mouse model and in turn superior transgene expression in the tumor. By small hairpin RNA for VEGF silencing, the c-polyplexes exerted significant tumor growth inhibition following with low systemic toxicity in the mouse. This study highlights the design of clickable polycations to construct crosslinked cationic nanopolyplexes for intravenous gene delivery against cancer.

Super-resolution Imaging of Structure, Molecular Composition, and Stability of Single Oligonucleotide Polyplexes.

The successful application of gene therapy relies on the development of safe and efficient delivery vectors. Cationic polymers such as cell-penetrating peptides (CPPs) can condense genetic material into nanoscale particles, called polyplexes, and induce cellular uptake. With respect to this point, several aspects of the nanoscale structure of polyplexes have remained elusive because of the difficulty in visualizing the molecular arrangement of the two components with nanometer resolution. This limitation has hampered the rational design of polyplexes based on direct structural information. Here, we used super-resolution imaging to study the structure and molecular composition of individual CPP-mRNA polyplexes with nanometer accuracy. We use two-color direct stochastic optical reconstruction microscopy (dSTORM) to unveil the impact of peptide stoichiometry on polyplex structure and composition and to assess their destabilization in blood serum. Our method provides information about the size and composition of individual polyplexes, allowing the study of such properties on a single polyplex basis. Furthermore, the differences in stoichiometry readily explain the differences in cellular uptake behavior. Thus, quantitative dSTORM of polyplexes is complementary to the currently used characterization techniques for understanding the determinants of polyplex activity in vitro and inside cells.

Novel Oligo-Guanidyl-PEG Carrier Forming Rod-Shaped Polyplexes.

A novel unconventional supramolecular oligo-cationic structure (Agm6-M-PEG-OCH3) has been synthesized to yield high efficiency therapeutic oligonucleotide (ON) delivery. Agm6-M-PEG-OCH3 was obtained by a multistep protocol that included the conjugation of agmatine (Agm) moieties to maltotriose (M), which was further derivatized with one poly(ethylene glycol) (PEG) chain. Gel electrophoresis analysis showed that the 19 base pairs dsDNA model ON completely associates with Agm6-M-PEG-OCH3 at 3 N/P molar ratio, which is in agreement with the in silico molecular predictions. Isothermal titration calorimetry (ITC) analyses showed that the Agm6-M-PEG-OCH3/ON association occurs through a combination of mechanisms depending on the N/P ratios resulting in different nanostructures. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the Agm6-M-PEG-OCH3/ON polyplexes have rod-shape structure with a mean diameter of 50-75 nm and aspect ratio depending on the N/P ratio. The polyplexes were stable over time in buffer, while a slight size increase was observed in the presence of serum proteins. Cell culture studies showed that neither Agm6-M-PEG-OCH3 nor polyplexes displayed cytotoxic effects. Cellular uptake depended on the cell line and polyplex composition: cellular internalization was higher in the case of MCF-7 and KB cells compared to MC3T3-E1 cells and polyplexes with smaller aspect ratio were taken-up by cells more efficiently than polyplexes with higher aspect ratio. Finally, preliminary studies showed that our novel carrier efficiently delivered ONs into cells providing gene silencing.

Poly(ethylene glycol)- block-poly(2-aminoethyl methacrylate hydrochloride)-Based Polyplexes as Serum-Tolerant Nanosystems for Enhanced Gene Delivery.

Incorporation of poly(ethylene glycol) (PEG) into polyplexes has been used as a promising approach to enhance their stability and reduce unwanted interactions with biomolecules. However, this strategy generally has a negative influence on cellular uptake and, consequently, on transfection of target cells. In this work, we explore the effect of PEGylation on biological and physicochemical properties of poly(2-aminoethyl methacrylate) (PAMA)-based polyplexes. For this purpose, different tailor-made PEG- b-PAMA block copolymers, and the respective homopolymers, were synthesized using the controlled/"living" radical polymerization method based on activators regenerated by electron transfer atom transfer radical polymerization. The obtained data show that PEG- b-PAMA-based polyplexes exhibited a much better transfection activity/cytotoxicity relationship than the corresponding non-PEGylated nanocarriers. The best formulation, prepared with the largest block copolymer (PEG45- b-PAMA168) at a 25:1 N/P ratio, presented a 350-fold higher transfection activity in the presence of serum than that obtained with polyplexes generated with the gold standard bPEI. This higher transfection activity was associated to an improved capability to overcome the intracellular barriers, namely the release from the endolysosomal pathway and the vector unpacking and consequent DNA release from the nanosystem inside cells. Moreover, these nanocarriers exhibit suitable physicochemical properties for gene delivery, namely reduced sizes, high DNA protection, and colloidal stability. Overall, these findings demonstrate the high potential of the PEG45- b-PAMA168 block copolymer as a gene delivery system.

Tunable Properties of Poly-DL-Lactide-Monomethoxypolyethylene Glycol Porous Microparticles for Sustained Release of Polyethylenimine-DNA Polyplexes.

Direct pulmonary delivery is a promising step in developing effective gene therapies for respiratory disease. Gene therapies can be used to treat the root cause of diseases, rather than just the symptoms. However, developing effective therapies that do not cause toxicity and that successfully reach the target site at therapeutic levels is challenging. We have developed a polymer-DNA complex utilizing polyethylene imine (PEI) and DNA, which was then encapsulated into poly(lactic acid)-co-monomethoxy poly(ethylene glycol) (PLA-mPEG) microparticles via double emulsion, solvent evaporation. Then, the resultant particle size, porosity, and encapsulation efficiency were measured as a function of altering preparation parameters. Microsphere formation was confirmed from scanning electron micrographs and the aerodynamic particle diameter was measured using an aerodynamic particle sizer. Several formulations produced particles with aerodynamic diameters in the 0-5 µm range despite having larger particle diameters which is indicative of porous particles. Furthermore, these aerodynamic diameters correspond to high deposition within the airways when inhaled and the measured DNA content indicated high encapsulation efficiency. Thus, this formulation provides promise for developing inhalable gene therapies.

Amphiphilic poly(α)glutamate polymeric micelles for systemic administration of siRNA to tumors.

RNAi therapeutics carried a great promise to the area of personalized medicine: the ability to target "undruggable" oncogenic pathways. Nevertheless, their efficient tumor targeting via systemic administration had not been resolved yet. Amphiphilic alkylated poly(α)glutamate amine (APA) can serve as a cationic carrier to the negatively-charged oligonucleotides. APA polymers complexed with siRNA to form round-shaped, homogenous and reproducible nano-sized polyplexes bearing ~50 nm size and slightly negative charge. In addition, APA:siRNA polyplexes were shown to be potent gene regulators in vitro. In light of these preferred physico-chemical characteristics, their performance as systemically-administered siRNA nanocarriers was investigated. Intravenously-injected APA:siRNA polyplexes accumulated selectively in tumors and did not accumulate in the lungs, heart, liver or spleen. Nevertheless, the polyplexes failed to induce specific mRNA degradation, hence neither reduction in tumor volume nor prolonged mice survival was seen.

Synthesis, characterization and evaluation of transfection efficiency of dexamethasone conjugated poly(propyleneimine) nanocarriers for gene delivery#.

CONTEXT: Polypropylenimine (PPI), a cationic dendrimer with defined structure and positive surface charge, is a potent non-viral vector. Dexamethasone (Dexa) conveys to the nucleus through interaction with its intracellular receptor. OBJECTIVE: This study develops efficient and non-toxic gene carriers through conjugation of Dexa at various percentages (5, 10 and 20%) to the fourth and the fifth generation PPIs (PPIG4s and PPIG5s). MATERIALS AND METHODS: The 21-OH group of Dexa (0.536 mmol) was modified with methanesulfonyl chloride (0.644 mmol) to activate it (Dexa-mesylate), and then it was conjugated to PPIs using Traut's reagent. After dialysis (48 h) and lyophilization, the physicochemical characteristics of products (PPI-Dexa) including zeta potential, size, buffering capacity and DNA condensing capability were investigated and compared with unmodified PPIs. Moreover, the cytotoxicity and transfection activity of the Dexa-modified PPIs were assessed using Neuro2A cells. RESULTS: Transfection of PPIG4 was close to PEI 25 kDa. Although the addition of Dexa to PPIG4s did not improve their transfection, their cytotoxicity was improved; especially in the carrier to DNA weight ratios (C/P) of one and two. The Dexa conjugation to PPIG5s enhanced their transfection at C/P ratio of one in both 5% (1.3-fold) and 10% (1.6-fold) Dexa grafting, of which the best result was observed in PPIG5-Dexa 10% at C/P ratio of one. DISCUSSION AND CONCLUSIONS: The modification of PPIs with Dexa is a promising approach to improve their cytotoxicity and transfection. The higher optimization of physicochemical characteristics, the better cell transfection and toxicity will be achieved.

A Poly(Propyleneimine) Dendrimer-Based Polyplex-System for Single-Chain Antibody-Mediated Targeted Delivery and Cellular Uptake of SiRNA.

Therapeutics based on small interfering RNAs (siRNAs) offer a great potential to treat so far incurable diseases or metastatic cancer. However, the broad application of siRNAs using various nonviral carrier systems is hampered by unspecific toxic side effects, poor pharmacokinetics due to unwanted delivery of siRNA-loaded nanoparticles into nontarget organs, or rapid renal excretion. In order to overcome these obstacles, several targeting strategies using chemically linked antibodies and ligands have emerged. This study reports a new modular polyplex carrier system for targeted delivery of siRNA, which is based on transfection-disabled maltose-modified poly(propyleneimine)-dendrimers (mal-PPI) bioconjugated to single chain fragment variables (scFvs). To achieve targeted delivery into tumor cells expressing the epidermal growth factor receptor variant III (EGFRvIII), monobiotinylated anti-EGFRvIII scFv fused to a Propionibacterium shermanii transcarboxylase-derived biotinylation acceptor (P-BAP) is bioconjugated to mal-PPI through a novel coupling strategy solely based on biotin-neutravidin bridging. In contrast to polyplexes containing an unspecific control scFv-P-BAP, the generated EGFRvIII-specific polyplexes are able to exclusively deliver siRNA to tumor cells and tumors by receptor-mediated endocytosis. These results suggest that receptor-mediated uptake of otherwise noninternalized mal-PPI-based polyplexes is a promising avenue to improve siRNA therapy of cancer, and introduce a novel strategy for modular bioconjugation of protein ligands to nanoparticles.

Dual-Function Polymeric HPMA Prodrugs for the Delivery of miRNA.

An HPMA-based polymeric prodrug of a CXCR4 antagonist, AMD3465 (P-SS-AMD), was developed as a dual-function carrier of therapeutic miRNA. P-SS-AMD was synthesized by a copolymerization of HPMA with a methacrylamide monomer in which the AMD3465 was attached via a self-immolative disulfide linker. P-SS-AMD showed effective release of the parent AMD3465 drug following treatment with intracellular levels of glutathione (GSH). The AMD3465 was released in the cells and exhibited functional CXCR4 antagonism, demonstrated by inhibition of the CXCR4-mediated cancer cell invasion. Due to its cationic character, P-SS-AMD could form polyplexes with miRNA and mediate efficient transfection of miR-200c mimics to downregulate expression of a downstream target ZEB-1 in cancer cells. The combined P-SS-AMD/miR-200c polyplexes showed improved ability to inhibit cancer cell migration when compared with individual treatments. The reported findings validate P-SS-AMD as a dual-function delivery vector that can simultaneously deliver a therapeutic miRNA and function as a polymeric prodrug of CXCR4 antagonist.