Smart polymeric carriers for enhanced intracellular delivery of therapeutic macromolecules

Limited cytoplasmic delivery of enzyme-susceptible drugs remains a significant challenge facing the development of protein and nucleic acid therapies that act in intracellular compartments. Researchers have examined several approaches, including fusogenic proteins and protein transduction domains, to enhance the intracellular delivery of the therapeutic cargo. This review summarises efforts to develop 'smart' pH-sensitive and membrane-destabilising polymers that can shuttle therapeutic peptide, protein and nucleic acid molecules past the endosomal membrane into the cytoplasm of targeted cells. Several classes of 'smart' non-degradable polymeric carriers have been developed that have proved effective both in vitro and in vivo in enhancing the cytoplasmic delivery of a variety of therapeutic molecules.     

pH-sensitive polymers that enhance intracellular drug delivery in vivo.

Cytosolic delivery from endosomes is critical for those drugs that are susceptible to attack by lysosomal enzymes, such as DNA, RNA, oligonucleotides, proteins and peptides. Therefore, we have designed pH-sensitive, membrane-disruptive polymers to enhance the release of drugs from the acidic endosomal compartment to the cytoplasm. We have found that one polymer in particular, poly(propylacrylic acid) (PPAA), is very effective at membrane disruption at pHs below 6.5, based on hemolysis studies. PPAA also significantly enhances in vitro transfections of lipoplex formulations in cell culture, and does so in the presence of as much as 50% serum. In this study, we have extended our in vitro hemolysis and cell culture studies to an in vivo murine excisional wound healing model. A pilot study with a green fluorescent protein (GFP)-encoding plasmid indicated that injection of formulations containing PPAA into healing wounds resulted in increased GFP expression. Subsequently, by administering sense and antisense DNA for the angiogenesis inhibitor thrombospondin-2 (TSP2), we were able to alter the wound healing response in TSP2-null and wild type mice, respectively. Our findings showed that when PPAA was added to lipoplex formulations, expression of TSP2 was enhanced in TSP2-null mice compared to control formulations. These results show that PPAA can enhance in vivo transfections and that inhibition of TSP2 expression may lead to improved wound healing. These results suggest that PPAA can provide significant improvements in the in vivo efficacy of drugs such as DNA.     

A new pH-responsive and glutathione-reactive, endosomal membrane-disruptive polymeric carrier for intracellular delivery of biomolecular drugs.

In this study, we have designed, synthesized and characterized a novel pH-responsive polymeric carrier for the enhanced cytoplasmic delivery of enzyme susceptible drugs, such as antisense oligonucleotides, proteins and peptides. A novel functionalized monomer, pyridyl disulfide acrylate, was synthesized and incorporated into an amphiphilic copolymer consisting of methacrylic acid and butyl acrylate, which resulted in a glutathione- and pH-sensitive, membrane-disruptive terpolymer with functional groups, that allow thiol-containing molecules to be readily conjugated. Conjugation and/or ionic complexation with oligopeptides or antisense oligonucleotides were performed and characterized. Hemolytic activity at low pHs remained high even after the conjugation/complexation with oligopeptides and asODNs. This polymer showed no toxicity, as determined with mouse 3T3 fibroblasts and human THP-1 macrophage-like cells. Uptake of the radiolabeled polymer and enhanced cytoplasmic delivery of FITC-ODN was also studied in THP-1 macrophage-like cells.     

Polymeric micelles with a pH-responsive structure as intracellular drug carriers.

Polymeric micelles based on poly(L-lactide)-b-poly(2-ethyl-2-oxazoline)-b-poly(L-lactide) (PLLA-PEOz-PLLA) ABA triblock copolymers were designed as intracellular drug carriers. The PLLA-PEOz-PLLA micelles adopt a "flower-like" arrangement with A-blocks at the core and a B-block on the shell under neutral condition. The deformation of the core-shell structure is then promoted by the aggregation of PEOzs due to the formation of inter- and intra-hydrogen bonding between protonated nitrogen and carbonyl groups. The experiments on in vitro release have confirmed that the release of doxorubicin (DOX) from micelles was successfully inhibited at pH 7.4. In contrast, an accelerated release of DOX from micelles was observed at acidic conditions. The results of growth inhibition assay indicated that the cell-killing rate of DOX-loaded micelles gradually approached that of free DOX as increasing the concentration and the incubation time. The overlay of fluorescent images on CLSM observation clearly demonstrated the colocalization of DOX with acidic compartments, suggesting that the drug release was successfully triggered in the acidic organelles by means of micelle deformation.     

Cellular uptake and intracellular fate of antisense oligonucleotides

Antisense oligonucleotides and short interfering RNAs are routinely used for gene function analysis and are being developed for clinical applications. The mechanism underlying internalization of free oligonucleotides into cells is poorly understood and inefficient in most cases. Antisense oligonucleotide delivery into ex vivo cells is routinely improved by the addition of cationic lipids. New chemical modifications and vectors allowing improved cellular delivery in vivo are being developed.     

Self-assembled and pH-responsive polymeric nanomicelles impart effective delivery of paclitaxel to cancer cells

Chemotherapy is an essential component of breast cancer therapy, but it is associated with serious side effects. Herein, a pluronic F68-based pH-responsive, and self-assembled nanomicelle system was designed to improve the delivery of paclitaxel (PTX) to breast cancer cells. Two pH-responsive pluronic F68-PTX conjugates i.e. succinoyl-linked conjugate (F68-SA-PTX) and cis-aconityl-linked conjugate (F68-CAA-PTX) were designed to respond the varying pH-environment in tumour tissue. Although both the linkers showed pH-sensitivity, the F68-CAA-PTX exhibited superior pH-sensitivity over the F68-SA-PTX and achieved a more selective release of PTX from the self-assembled nanomicelles. The prepared nanomicelles were characterized by dynamic light scattering, transmittance electron microscopy, differential scanning calorimetry and powder X-ray diffraction techniques. The anticancer activity of prepared nanomicelles and pure PTX were evaluated by 2D cytotoxicity assay against breast cancer cell line MDA-MB-231 and in the real tumour environments i.e. 3D tumor spheroids of MDA-MB-231 cells. The highest cytotoxicity effect of PTX was observed with F68-CAA-PTX nanomicelles followed by F68-SA-PTX and free PTX. Further, the F68-CAA-PTX nanomicelles also induced significant apoptosis with a combination of increase in ROS generation, decrease in the depolarisation of MMP and G2/M cell cycle arrest. These observed results provide a new insight for breast cancer treatment using pluronic nanomicelles.

Self-assembled and pH-responsive polymeric nanomicelles were prepared for the delivery of paclitaxel to cancer cells.     

Effective intracellular delivery of oligonucleotides in order to make sense of antisense.

For more than two decades, antisense oligonucleotides (ODNs) have been used to modulate gene expression for the purpose of applications in cell biology and for development of novel sophisticated medical therapeutics. Conceptually, the antisense approach represents an elegant strategy, involving the targeting to and association of an ODN sequence with a specific mRNA via base-pairing, resulting in an impairment of functional and/or harmful protein expression in normal and diseased cells/tissue, respectively. Apart from ODN stability, its efficiency very much depends on intracellular delivery and release/access to the target side, issues that are still relatively poorly understood. Since free ODNs enter cells relatively poorly, appropriate carriers, often composed of polymers and cationic lipids, have been developed. Such carriers allow efficient delivery of ODNs into cells in vitro, and the mechanisms of delivery, both in terms of biophysical requirements for the carrier and cell biological features of uptake, are gradually becoming apparent. To become effective, ODNs require delivery into the nucleus, which necessitates release of internalized ODNs from endosomal compartments, an event that seems to depend on the nature of the delivery vehicle and distinct structural shape changes. Interestingly, evidence is accumulating which suggests that by modulating the surface properties of the carrier, the kinetics of such changes can be controlled, thus providing possibilities for programmable release of the carrier contents. Here, consideration will also be given to antisense design and chemistry, and the challenge of extra- and intracellular barriers to be overcome in the delivery process.     

In vitro and in vivo intracellular liposomal delivery of antisense oligonucleotides and anticancer drug.

The specific aims of this investigation were (1) to show that conventional and PEGylated liposomes can penetrate cancer cells in vitro and in vivo; (2) to demonstrate that liposomes can be successfully used both for cytoplasmic and nuclear delivery of therapeutics, including anticancer drugs and antisense oligonucleotides; (3) to examine the specific activity of anticancer drugs and nucleotides delivered inside tumor cells by PEGylated liposomes; and (4) to confirm that simultaneous inhibition of pump and nonpump cellular resistance by liposomal ASO can substantially enhance the antitumor activity of traditional well established anticancer drugs in mice bearing xenografts of human multidrug resistant ovarian carcinoma. Experimental results show that PEGylated liposomes are capable of penetrating directly into tumor cells after systemic administration in vivo and do successfully provide cytoplasmic and nuclear delivery of encapsulated anticancer drug (doxorubicin, DOX) and antisense oligonucleotides (ASO). Encapsulation of DOX and ASO into liposomes substantially increased their specific activity. Simultaneous suppression of pump and nonpump resistance dramatically enhanced the ability of DOX for inducing apoptosis leading to higher in vitro cytotoxicity and in vivo antitumor activity.     

Innovative nanotechnologies for the delivery of oligonucleotides and siRNA.

One way to reach intracellular therapeutic targets in cells consists in the use of short nucleic acids which will bind specifically to on targets thanks to either Watson–Crick base pairing or protein nucleic acids recognition rules. Among these short nucleic acids an important class of therapeutic agents is antisense oligonucleotides and siRNAs. However, the major problem of nucleic acids is their poor stability in biological media. One method, among others, to solve the stability problem is the use of colloïdal carriers such as nanoparticles. Nanoparticles have already been applied with success to in vitro drug delivery to particular types of cells and in vivo in several experimental models. Many membrane and intracellular processes deal with nanosized structure (typically 100 nm) which are processed further through the recognition sites of receptors and enzymes. Thus non-viral nanoparticles are interesting candidates to present biochemical molecules such as nucleic acids and proteins to cells as well as to protect them in vivo during delivery. This review focuses on the recent developments in the design of nanotechnologies to improve the delivery of antisense oligonucleotides and siRNAs.     

Targeted delivery of antisense oligonucleotides in cancer.

Formulations of antisense oligonucleotides (asODNs) against c-myb or c-myc protooncogenes have been prepared by a new technique that sequesters cationic lipid in the interior of a lipid particle. This technique results in high loading efficiency for the asODNs, small particle size and good stability. When targeted against melanoma cells or neuroblastoma cells via anti-GD(2) coupled at the particle surface, increased cell binding to the cells could be demonstrated. Targeted formulations showed greater inhibition of cell proliferation compared to non-targeted formulations or free drug. Inhibition of cell proliferation was demonstrated to be due to down-regulation of c-myb or c-myc protein expression. The formulations have long-circulation times in vivo, and evaluation for in vivo antitumor activity is currently underway.     

Mechanisms of co-modified liver-targeting liposomes as gene delivery carriers based on cellular uptake and antigens inhibition effect.

In order to deliver antisense oligonucleotides (asODN) into hepatocytes orientedly in the treatment of hepatitis B virus (HBV) infection, the liver-targeting cationic liposomes was developed as a gene carrier, which was co-modified with the ligand of the asialoglycoprotein receptor (ASGPR), beta-sitosterol-beta-d-glucoside (sito-G) and the nonionic surfactant, Brij 35. Flow cytometry (FCM) analysis and enzyme-linked immunosorbent assay (ELISA) showed that the asODN-encapsulating cationic liposomes exhibited high transfection efficiency and strong antigens inhibition effect in primary rat hepatocytes and HepG2.2.15 cells, respectively. With the help of several inhibitors acting on different steps during the targeting lipofection, the cellular uptake mechanisms of the co-modified liver-targeting cationic liposomes were investigated through antigens inhibition effect assay and confocal laser scanning microscopy (CLSM) analysis. The cellular uptake with high transfection efficiency seemed to involve both endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance ASGPR-mediated endocytosis, the nonionic surfactant Brij 35 seemed to be able to facilitate membrane fusion, and the co-modification resulted in the most efficient transfection but no enhanced cytotoxicity. These results suggested that the co-modified liver-targeting cationic liposomes would be a specific and effective carrier to transfer asODN into hepatocytes infected with HBV orientedly.     

On the biological activity of anti-ICAM-1 oligonucleotides complexed to non-viral carriers.

An important challenge in antisense technology remains the adequate delivery of the oligonucleotides (ON) to individual cells. Understanding the subcellular distribution of ONs and their carrier is essential to explain the (lack of) biological activity. The ability of several cationic carriers to efficiently deliver anti-ICAM-1 oligonucleotides to their site of action was studied using a cell-based assay. In this assay we evaluated the ability of the ONs to downregulate the expression of the ICAM-1-protein in A549 cells. To understand why some carrier/ONs combinations showed biological activity while others failed, flow cytometry and confocal laser scanning microscopy (CLSM) measurements were used to study cellular uptake and intracellular distribution of the (fluorescently labeled) ONs. We showed that free ONs (both PS-ONs and PO-ONs) and ONs complexed to pEGpEI failed to decrease the ICAM-1 protein level. This was due to the inability of the (free or complexed) ONs to enter the cell, as shown by flow cytometry and CLSM. Flow cytometry and CLSM showed cellular uptake when PO-ONs and PS-ONs were complexed to graft-pDMAEMA and Lipofectin. However, while the uptake and intracellular localization seemed similar for ONs complexed to, respectively, graft-pDMAEMA and Lipofectin, the biological activity of the ONs was clearly dependent on their carrier: both PO-ONs and PS-ONs complexed to graft-pDMAEMA reduced the ICAM-1 expression; however, when complexed to Lipofectin only PS-ONs showed biological activity. Also, PS-ONs complexed to graft-pDMAEMA were more active than PO-ONs complexed to graft-pDMAEMA which could not be explained by the results from CLSM and flow cytometry. While the ICAM-1 assay proves whether a certain pharmaceutical carrier successfully delivers ONs or not, it does not answer the important question why one carrier is successful while another one fails. Also, our study shows that flow cytometry and CLSM, although useful techniques, failed to clearly explain the difference in transfection behavior between graft-pDMAEMA and Lipofectin. As ONs become susceptible to degradation by cytosolic DNase as soon as they are released from their carrier, one could argue that a better understanding of the time and (intracellular) place at which the dissociation of the complexes occurs could be crucial to fully explain our observations.     

Overview of target validation and the impact of oligonucleotides

During target validation, researchers attempt to modulate the activity of potential drug targets in relevant cell and/or animal disease models in order to identify those that might be expected to have therapeutic benefit in human patients. This has become increasingly important with the large expansion of potential targets identified by the human genome project and as a result of the spiralling costs of bringing drugs to market. This review will present an overview of the target-validation mechanism and examine the strengths and weaknesses of using oligonucleotide-based technologies such as antisense, short interfering RNA and aptamers.     

Pulmonary delivery of insulin by liposomal carriers.

Growing attention has been given to the potential of a pulmonary route as a non-invasive administration for systemic delivery of therapeutic agents (mainly peptides and proteins). The lungs provide a large absorptive surface area, extremely thin absorptive mucosal membrane, and good blood supply. The non-invasive nature of this pathway makes it especially valuable for the delivery of large molecular protein. However, pulmonary delivery of peptides and proteins is complicated by the complexity of the anatomic structure of the human respiratory system and the effect of disposition exerted by the respiration process. In this study, novel nebulizer-compatible liposomal carrier for aerosol pulmonary drug delivery of insulin was developed and characterized. Experimental results showed that insulin could be efficiently encapsulated into liposomes by preformed vesicles and detergent dialyzing method. The optimal encapsulation efficiency was achieved when 40% ethanol was used. The particle size of liposomal aerosols from ultrasonic nebulizer approximated to 1 mum. Insulin was stable in the liposomal solution. Animal studies showed that plasma glucose level was effectively reduced when liposomal insulin was delivered by inhalation route of using aerosolized insulin-encapsulated liposomes. Including fluorescent probe (phosphatidylethanolamine-rhodamine) into liposome, we found that the liposomal carriers were effectively and homogeneously distributed in the lung aveolar. Liposome-mediated pulmonary drug delivery promotes an increase in drug retention-time in the lungs, and more importantly, a reduction in extrapulmonary side-effects which invariably results in enhanced therapeutic efficacies.     

Aspects of the transport and delivery of antisense oligonucleotides

This article will examine recent developments concerning the cellular uptake and subcellular trafficking of antisense oligonucleotides. It will also examine the merits of various delivery strategies for oligonucleotides. The use of conjugates of oligonucleotides with 'cell penetrating peptides' as a promising delivery technology will be emphasized.     

Fabrication and characterization of structurally stable pH-responsive polymeric vesicles by polymerization-induced self-assembly

Smart polymeric vesicles with both tertiary amine and epoxy functional groups were fabricated for the first time via a reversible addition–fragmentation chain transfer dispersion polymerization approach, using (2-(diisopropylamino)ethyl methacrylate (DIPEMA) and glycidyl methacrylate (GlyMA) in an ethanol–water mixture. Monitoring of the in situ polymerization revealed the low molecular weight distributions and the intermediate structures of spheres and worms, indicating an evolution in particle morphology. A phase diagram was constructed for reproducible fabrication of the vesicles, and copolymer composition was found to be more related to particle morphology. The vesicles exhibited superior structural stability for the cross-linking of the core through epoxydiamine chemistry, and intelligent pH responsibility due to the presence of the tertiary amine groups. The cross-linked vesicles showed good stability and reversibility during the swelling and shrinking cycles by switching the pH values, which endowed them with potential cell-like transmission functions. This research thus provides a method for producing structurally stable pH-responsive polymeric vesicles, and the reported vesicles are based on commercially available starting materials for possible industrial scale-up.

Smart polymeric vesicles with both tertiary amine and epoxy functional groups were fabricated for the first time via a reversible addition–fragmentation chain transfer dispersion polymerization approach.     

Thermosensitive and biodegradable polymeric micelles for paclitaxel delivery.

The preparation, release and in vitro cytotoxicity of a novel polymeric micellar formulation of paclitaxel (PTX) were investigated. The micelles consisted of an AB block copolymer of poly(N-(2-hydroxypropyl) methacrylamide lactate) and poly(ethylene glycol) (pHPMAmDL-b-PEG). Taking advantage of the thermosensitivity of pHPMAmDL-b-PEG, the loading was done by simply mixing of a small volume of a concentrated PTX solution in ethanol and an aqueous polymer solution and subsequent heating of the resulting solution above the critical micelle temperature of the polymer. PTX could be almost quantitatively loaded in the micelles up to 2 mg/mL. By dynamic light scattering and cryo-transmission electron microscopy, it was shown that PTX-loaded micelles have a mean size around 60 nm with narrow size distribution. At pH 8.8 and 37 degrees C, PTX-loaded micelles destabilized within 10 h due to the hydrolysis of the lactic acid side group of the pHPMAmDL. Because the hydrolysis of the lactic acid side groups is first order in hydroxyl ion concentration, the micelles were stable for about 200 h at physiological conditions. The presence of serum proteins did not have an adverse effect on the stability of the micelles during at least 15 h. Interestingly, the dissolution kinetics of pHPMAmDL-b-PEG micelles was retarded by incorporation of PTX, indicating a strong interaction between PTX and the pHPMAmDL block. The PTX-loaded micelles showed a release of the incorporated 70% of PTX during 20 h at 37 degrees C and at pH 7.4. PTX-loaded pHPMAmDL-b-PEG micelles showed comparable in vitro cytotoxicity against B16F10 cells compared to the Taxol standard formulation containing Cremophor EL, while pHPMAmDL-b-PEG micelles without PTX were far less toxic than the Cremophor EL vehicle. Confocal laser-scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) analysis of fluorescently labelled micelles showed that pHPMAmDL-b-PEG micelles were internalized by the B16F10 cells. The present results suggest that pHPMAmDL-b-PEG block copolymer micelles are a promising delivery system for the parenteral administration of PTX.     

Topical and transdermal delivery of antisense oligonucleotides

Antisense oligonucleotides have great potential as therapeutic agents. As a result, a variety of chemistries have been developed to improve the efficacy of these molecules without compromising their specificity. Because the skin is such a large organ with extensive accessibility, it is a natural target for drug delivery. It is, however, an effective barrier, so physical and chemical methods to improve drug penetration have been developed. These enhancement techniques can be combined with modified oligonucleotide chemistries to provide sufficient levels of antisense activity either within the skin or systemically. This review will describe in vitro and in vivo experiments that demonstrate the potential of antisense oligonucleotides to treat both dermal and systemic disorders.