pH-responsive poly(styrene-alt-maleic anhydride) alkylamide copolymers for intracellular drug delivery

Many macromolecular therapeutics such as peptides, proteins, antisense oligodeoxynucleotides (ASODN), and short interfering RNA (siRNA) are active only in the cytoplasm or nucleus of targeted cells. Endocytosis is the primary route for cellular uptake of these molecules, which results in their accumulation in the endosomal-lysosomal trafficking pathway and loss of therapeutic activity. In this article, we describe the synthesis and pH-dependent membrane-destabilizing activity of a new "smart" polymer family that can be utilized to enhance the intracellular delivery of therapeutic macromolecules through the endosomal membrane barrier into the cytoplasm of targeted cells. These polymers are propylamine, butylamine, and pentylamine derivatives of poly(styrene-alt-maleic anhydride) (PSMA) copolymers. The PSMA-alkylamide derivatives are hydrophilic and membrane-inactive at physiological pH; however, they become hydrophobic and membrane-disruptive in response to endosomal pH values as measured by their hemolytic activity. Results show that the pH-dependent membrane-destabilizing activity of PSMA derivatives can be controlled by varying the length of the alkylamine group, the degree of modification of the copolymer, and the molecular weight of the PSMA copolymer backbone. Butylamine and pentylamine derivatives of PSMA copolymers exhibited more than 80% hemolysis at endosomal pH values, which suggests their potential as a platform of "smart" polymeric carriers for enhanced cytoplasmic delivery of a variety of therapeutic macromolecules.     

Kinetically controlled cellular interactions of polymer-polymer and polymer-liposome nanohybrid systems

Although bioactive polymers such as cationic polymers have demonstrated potential as drug carriers and nonviral gene delivery vectors, high toxicity and uncontrolled, instantaneous cellular interactions of those vectors have hindered the successful implementation In Vivo. Fine control over the cellular interactions of a potential drug/gene delivery vector would be thus desirable. Herein, we have designed nanohybrid systems (100-150 nm in diameter) that combine the polycations with protective outer layers consisting of biodegradable polymeric nanoparticles (NPs) or liposomes. A commonly used polycation polyethylenimine (PEI) was employed after conjugation with rhodamine (RITC). The PEI-RITC conjugates were then encapsulated into (i) polymeric NPs made of either poly(lactide-co-glycolide) (PLGA) or poly(ethylene glycol)-b-poly(lactide-co-glycolide) (PEG-PLGA); or (ii) PEGylated liposomes, resulting in three nanohybrid systems. Through the nanohybridization, both cellular uptake and cytotoxicity of the nanohybrids were kinetically controlled. The cytotoxicity assay using MCF-7 cells revealed that liposome-based nanohybrids exhibited the least toxicity, followed by PEG-PLGA- and PLGA-based NPs after 24 h incubation. The different kinetics of cellular uptake was also observed, the liposome-based systems being the fastest and PLGA-based systems being the slowest. The results present a potential delivery platform with enhanced control over its biological interaction kinetics and passive targeting capability through size control.     

Enhanced delivery of cell-penetrating peptide-peptide nucleic acid conjugates by endosomal disruption

Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery.     

Receptor-targeted nanocarriers for therapeutic delivery to cancer

Efficient and site-specific delivery of therapeutic drugs is a critical challenge in clinical treatment of cancer. Nano-sized carriers such as liposomes, micelles, and polymeric nanoparticles have been investigated for improving bioavailability and pharmacokinetic properties of therapeutics via various mechanisms, for example, the enhanced permeability and retention (EPR) effect. Further improvement can potentially be achieved by conjugation of targeting ligands onto nanocarriers to achieve selective delivery to the tumour cell or the tumour vasculature. Indeed, receptor-targeted nanocarrier delivery has been shown to improve therapeutic responses both in vitro and in vivo. A variety of ligands have been investigated including folate, transferrin, antibodies, peptides and aptamers. Multiple functionalities can be incorporated into the design of nanoparticles, e.g., to enable imaging and triggered intracellular drug release. In this review, we mainly focus on recent advances on the development of targeted nanocarriers and will introduce novel concepts such as multi-targeting and multi-functional nanoparticles.     

Direct translocation as major cellular uptake for CADY self-assembling peptide-based nanoparticles

Cell penetrating peptides constitute a potent approach to overcome the limitations of in vivo siRNA delivery. We recently proposed a peptide-based nanoparticle system, CADY, for efficient delivery of siRNA into numerous cell lines. CADY is a secondary amphipathic peptide that forms stable complexes with siRNA thereby improving both their cellular uptake and biological response. With the aim of understanding the cellular uptake mechanism of CADY:siRNA complexes, we have combined biochemical, confocal and electron microscopy approaches. In the present work, we provide evidence that the major route for CADY:siRNA cellular uptake involves direct translocation through the membrane but not the endosomal pathway. We have demonstrated that CADY:siRNA complexes do not colocalize with most endosomal markers and remain fully active in the presence of inhibitors of the endosomal pathway. Moreover, neither electrostatic interactions with cell surface heparan sulphates nor membrane potential are essential for CADY:siRNA cell entry. In contrast, we have shown that CADY:siRNA complexes clearly induce a transient cell membrane permeabilization, which is rapidly restored by cell membrane fluidity. Therefore, we propose that direct translocation is the major gate for cell entry of CADY:siRNA complexes. Membrane perturbation and uptake are driven mainly by the ability of CADY to interact with phospholipids within the cell membrane, followed by rapid localization of the complex in the cytoplasm, without affecting cell integrity or viability.     

Receptor-targeted nanocarriers for therapeutic delivery to cancer

Abstract

Efficient and site-specific delivery of therapeutic drugs is a critical challenge in clinical treatment of cancer. Nano-sized carriers such as liposomes, micelles, and polymeric nanoparticles have been investigated for improving bioavailability and pharmacokinetic properties of therapeutics via various mechanisms, for example, the enhanced permeability and retention (EPR) effect. Further improvement can potentially be achieved by conjugation of targeting ligands onto nanocarriers to achieve selective delivery to the tumour cell or the tumour vasculature. Indeed, receptor-targeted nanocarrier delivery has been shown to improve therapeutic responses both in vitro and in vivo. A variety of ligands have been investigated including folate, transferrin, antibodies, peptides and aptamers. Multiple functionalities can be incorporated into the design of nanoparticles, e.g., to enable imaging and triggered intracellular drug release. In this review, we mainly focus on recent advances on the development of targeted nanocarriers and will introduce novel concepts such as multi-targeting and multi-functional nanoparticles.     

Effect of thiol pendant conjugates on plasmid DNA binding, release, and stability of polymeric delivery vectors

Polymers have attracted much attention as potential gene delivery vectors due to their chemical and structural versatility. However, several challenges associated with polymeric carriers, including low transfection efficiencies, insufficient cargo release, and high cytotoxicity levels have prevented clinical implementation. Strong electrostatic interactions between polymeric carriers and DNA cargo can prohibit complete cargo release within the cell. As a result, cargo DNA never reaches the cell's nucleus where gene expression takes place. In addition, highly charged cationic polymers have been correlated with high cytotoxicity levels, making them unsuitable carriers in vivo. Using poly(allylamine) (PAA) as a model, we investigated how pH-sensitive disulfide cross-linked polymer networks can improve the delivery potential of cationic polymer carriers. To accomplish this, we conjugated thiol-terminated pendant chains onto the primary amines of PAA using 2-iminothiolane, developing three new polymer vectors with 5, 13, or 20% thiol modification. Unmodified PAA and thiol-conjugated polymers were tested for their ability to bind and release plasmid DNA, their capacity to protect genetic cargo from enzymatic degradation, and their potential for endolysosomal escape. Our results demonstrate that polymer-plasmid complexes (polyplexes) formed by the 13% thiolated polymer demonstrate the greatest delivery potential. At high N/P ratios, all thiolated polymers (but not unmodified counterparts) were able to resist decomplexation in the presence of heparin, a negatively charged polysaccharide used to mimic in vivo polyplex-protein interactions. Further, all thiolated polymers exhibited higher buffering capacities than unmodified PAA and, therefore, have a greater potential for endolysosomal escape. However, 5 and 20% thiolated polymers exhibited poor DNA binding-release kinetics, making them unsuitable carriers for gene delivery. The 13% thiolated polymers, on the other hand, displayed high DNA binding efficiency and pH-sensitive release.     

Arginine-rich cross-linking peptides with different SV40 nuclear localization signal content as vectors for intranuclear DNA delivery

The major barriers for intracellular DNA transportation by cationic polymers are their toxicity, poor endosomal escape and inefficient nuclear uptake. Therefore, we designed novel modular peptide-based carriers modified with SV40 nuclear localization signal (NLS). Core peptide consists of arginine, histidine and cysteine residues for DNA condensation, endosomal escape promotion and interpeptide cross-linking, respectively. We investigated three polyplexes with different NLS content (10?mol%, 50?mol% and 90?mol% of SV40 NLS) as vectors for intranuclear DNA delivery. All carriers tested were able to condense DNA, to protect it from DNAase I and were not toxic to the cells. We observed that cell cycle arrest by hydroxyurea did not affect transfection efficacy of NLS-modified carriers which we confirmed using quantitative confocal microscopy analysis. Overall, peptide carrier modified with 90?mol% of SV40 NLS provided efficient transfection and nuclear uptake in non-dividing cells. Thus, incorporation of NLS into arginine-rich cross-linking peptides is an adequate approach to the development of efficient intranuclear gene delivery vehicles.     

Multifunctional synthetic poly(L-glutamic acid)-based cancer therapeutic and imaging agents

Modern polymer chemistry has led to the generation of a number of biocompatible synthetic polymers that have been increasingly studied as efficient carriers for drugs and imaging agents. Synthetic biocompatible polymers have been used to improve the efficacy of both small-molecular-weight therapeutics and imaging agents. Furthermore, multiple targeted anticancer agents and/or imaging reporters can be attached to a single polymer chain, allowing multifunctional and/or multimodality therapy and molecular imaging. Having both an anticancer drug and an imaging reporter in a single polymer chain allows noninvasive real-time visualization of the pharmacokinetics of polymeric drug delivery systems, which can uncover and explain the complicated mechanisms of in vivo drug delivery and their correlation to pharmacodynamics. This review examines the use of the synthetic biocompatible polymer poly(L-glutamic acid) (PG) as an efficient carrier of cancer therapeutics and imaging agents. This review summarizes and updates our recent research on the use of PG as a platform for drug delivery and molecular imaging, including recent clinical findings with respect to PG-paclitaxel (PG-TXL), the combination of PG-TXL with radiotherapy, mechanisms of action of PG-TXL, and noninvasive visualization of in vivo delivery of polymeric conjugates with contrast-enhanced magnetic resonance imaging, optical imaging, and multimodality imaging.     

Photochemically enhanced cellular delivery of cell penetrating peptide-PNA conjugates

Recent studies have shown that endosomal release is a major rate-limiting step for cellular delivery via a variety of cationic cell penetrating peptides. Thus, methods and/or protocols for effective release of endosomally entrapped drugs are highly warranted. Photochemical internalization (PCI) has previously been proposed for this purpose. Here, we demonstrate an enhancement of up to two orders of magnitude of the antisense effects (cytosolic/nuclear) of peptide nucleic acid-peptide conjugates (Tat, Arg7, KLA) in HeLa cells by a PCI approach using the photosensitizer AlPc2a. These results emphasize the importance of endosomal release for cellular activity of this type of drug delivery and also raise hope that methods like PCI which have applications for in vivo use may also enhance the bioavailability and in vivo efficacy of these types of conjugates.     

Non-phagocytic uptake of intravenously injected microspheres in rat spleen: influence of particle size and hydrophilic coating

A recent development in prolonging the circulation time of drug carriers, such as liposomes and microspheres, has been to minimize their removal by macrophages of the reticuloendothelial system by covering their surface with hydrophilic polymers such as poloxamers, poloxamines and poly(ethyleneglycols). Here we demonstrate that this strategy may not necessarily prolong the circulatory half-life of drug carriers in all animal models. In rats, as opposed to rabbits, a non-phagocytic mechanism in the spleen may be triggered to remove efficiently from the blood drug carriers coated with hydrophilic coatings. Both the size of particle and its hydrophilic coating may act synergistically to trigger this non-phagocytic mechanism. In rats, a remarkable log to log relationship between particle size and spleen uptake was observed for both uncoated and polymeric coated microspheres. The potential implication of these observations in site-specific delivery of drug carriers is discussed.     

Bioinspired polymers that control intracellular drug delivery

One of the important characteristics of biological systems is their ability to change important properties in response to small environmental signals. The molecular mechanisms that biological molecules utilize to sense and respond provide interesting models for the development of “smart” polymeric biomaterials with biomimetic properties. An important example of this is the protein coat of viruses, which contains peptide units that facilitate the trafficking of the virus into the cell via endocytosis, then out of the endosome into the cytoplasm, and from there into the nucleus. We have designed a family of synthetic polymers whose compositions have been designed to mimic specific peptides on viral coats that facilitate endosomal escape. Our biomimetic polymers are responsive to the lowered pH within endosomes, leading to disruption of the endosomal membrane and release of important biomolecular drugs such as DNA, RNA, peptides and proteins to the cytoplasm before they are trafficked to lysosomes and degraded by lysosomal enzymes. In this article, we review our work on the design, synthesis and action of such smart, pH-sensitive polymers.     

Prolonged gene silencing in hepatoma cells and primary hepatocytes after small interfering RNA delivery with biodegradable poly(beta-amino esters)

Background

Small interfering (si)RNA mediated inhibition of oncogenes or viral genes may offer great opportunities for the treatment of several diseases such as hepatocellular carcinoma and viral hepatitis. However, the development of siRNAs as therapeutic agents strongly depends on the availability of safe and effective intracellular delivery systems. Poly(β‐amino esters) (PbAEs) are, in contrast to many other cationic polymers evaluated in siRNA delivery, biodegradable into smaller, nontoxic molecules.

Methods and Results

We show for the first time that PbAE : siRNA complexes, containing 1,4‐butanediol (PbAE1) or 1,6‐hexanediol (PbAE2) diacrylate‐based polymers, induced efficient gene silencing in both hepatoma cells and primary hepatocytes without causing significant cytotoxicity. Furthermore, carriers that slowly release the siRNA into the cytoplasm and hence induce a prolonged gene silencing are of major clinical interest, especially in fast dividing tumour cells. Therefore, we also studied the duration of gene silencing in the hepatoma cells and found that it was maintained for at least 5 days after siRNA delivery with PbAE2, the polymer with the slowest degradation kinetics.

Conclusions

From the time‐dependent cellular distribution of these PbAE : siRNA complexes, we suggest that the slowly degrading PbAE2 causes a sustained endosomal release of siRNA during a much longer period than PbAE1. This may support the hypothesis that the endosomal release mechanism of PbAE : siRNA complexes is based on an increase of osmotic pressure in the endosomal vesicles after polymer hydrolysis. In conclusion, our results show that both PbAEs, and especially PbAE2, open up new perspectives for the development of efficient biodegradable siRNA carriers suitable for clinical applications. Copyright © 2008 John Wiley & Sons, Ltd.     

Endosomal escape of delivered mRNA from endosomal recycling tubules visualized at the nanoscale

Delivery of exogenous mRNA using lipid nanoparticles (LNPs) is a promising strategy for therapeutics. However, a bottleneck remains in the poor understanding of the parameters that correlate with endosomal escape versus cytotoxicity. To address this problem, we compared the endosomal distribution of six LNP-mRNA formulations of diverse chemical composition and efficacy, similar to those used in mRNA-based vaccines, in primary human adipocytes, fibroblasts, and HeLa cells. Surprisingly, we found that total uptake is not a sufficient predictor of delivery, and different LNPs vary considerably in endosomal distributions. Prolonged uptake impaired endosomal acidification, a sign of cytotoxicity, and caused mRNA to accumulate in compartments defective in cargo transport and unproductive for delivery. In contrast, early endocytic/recycling compartments have the highest probability for mRNA escape. By using super-resolution microscopy, we could resolve a single LNP-mRNA within subendosomal compartments and capture events of mRNA escape from endosomal recycling tubules. Our results change the view of the mechanisms of endosomal escape and define quantitative parameters to guide the development of mRNA formulations toward higher efficacy and lower cytotoxicity.     

Physicochemical properties of polymers: An important system to overcome the cell barriers in gene transfection

Delivery of the macromolecules including DNA, miRNA, and antisense oligonucleotides is typically mediated by carriers due to the large size and negative charge. Different physical (e.g., gene gun or electroporation), and chemical (e.g., cationic polymer or lipid) vectors have been already used to improve the efficiency of gene transfer. Polymer‐based DNA delivery systems have attracted special interest, in particular via intravenous injection with many intra‐ and extracellular barriers. The recent progress has shown that stimuli‐responsive polymers entitled as multifunctional nucleic acid vehicles can act to target specific cells. These nonviral carriers are classified by the type of stimulus including reduction potential, pH, and temperature. Generally, the physicochemical characterization of DNA‐polymer complexes is critical to enhance the transfection potency via protection of DNA from nuclease digestion, endosomal escape, and nuclear localization. The successful clinical applications will depend on an exact insight of barriers in gene delivery and development of carriers overcoming these barriers. Consequently, improvement of novel cationic polymers with low toxicity and effective for biomedical use has attracted a great attention in gene therapy. This article summarizes the main physicochemical and biological properties of polyplexes describing their gene transfection behavior, in vitro and in vivo. In this line, the relative efficiencies of various cationic polymers are compared. © 2015 Wiley Periodicals, Inc. Biopolymers 103: 363–375, 2015.     

Enhanced Cellular Uptake of Albumin-Based Lyophilisomes when Functionalized with Cell-Penetrating Peptide TAT in HeLa Cells

Lyophilisomes are a novel class of biodegradable proteinaceous nano/micrometer capsules with potential use as drug delivery carrier. Cell-penetrating peptides (CPPs) including the TAT peptide have been successfully implemented for intracellular delivery of a broad variety of cargos including various nanoparticulate pharmaceutical carriers. In the present study, lyophilisomes were modified using CPPs in order to achieve enhanced cellular uptake. Lyophilisomes were prepared by a freezing, annealing, and lyophilization method and a cystein-elongated TAT peptide was conjugated to the lyophilisomes using a heterobifunctional linker. Fluorescent-activated cell sorting (FACS) was utilized to acquire a lyophilisome population with a particle diameter smaller than 1000 nm. Cultured HeLa, OVCAR-3, Caco-2 and SKOV-3 cells were exposed to unmodified lyophilisomes and TAT-conjugated lyophilisomes and examined with FACS. HeLa cells were investigated in more detail using a trypan blue quenching assay, confocal microscopy, and transmission electron microscopy. TAT-conjugation strongly increased binding and cellular uptake of lyophilisomes in a time-dependent manner in vitro, as assessed by FACS. These results were confirmed by confocal microscopy. Transmission electron microscopy indicated rapid cellular uptake of TAT-conjugated lyophilisomes via phagocytosis and/or macropinocytosis. In conclusion, TAT-peptides conjugated to albumin-based lyophilisomes are able to enhance cellular uptake of lyophilisomes in HeLa cells.     

Characterization of Ku70(2)-NLS as bipartite nuclear localization sequence for non-viral gene delivery

Several barriers have to be overcome in order to achieve gene expression in target cells, e.g. cellular uptake, endosomal release and translocation to the nucleus. Nuclear localization sequences (NLS) enhance gene delivery by increasing the uptake of plasmid DNA (pDNA) to the nucleus. So far, only monopartite NLS were analysed for non-viral gene delivery. In this study, we examined the characteristics of a novel bipartite NLS like construct, namely NLS Ku70. We synthesized a dimeric structure of a modified NLS from the Ku70 protein (Ku70(2)-NLS), a nuclear transport active mutant of Ku70(2)-NLS (s1Ku70(2)-NLS) and a nuclear transport deficient mutant of Ku70(2)-NLS (s2Ku70(2)). We examined the transfection efficiency of binary Ku70(2)-NLS/DNA and ternary Ku70(2)-NLS/PEI/DNA gene vector complexes in vitro by using standard transfection protocols as well as the magnetofection method. The application of Ku70(2)-NLS and s1Ku70(2)-NLS increased gene transfer efficiency in vitro and in vivo. This study shows for the first time that the use of bipartite NLS compounds alone or in combination with cationic polymers is a promising strategy to enhance the efficiency of non-viral gene transfer.     

Conjugation of arginine-glycine-aspartic acid peptides to poly(ethylene oxide)-b-poly(epsilon-caprolactone) micelles for enhanced intracellular drug delivery to metastatic tumor cells

An arginine-glycine-aspartic acid (RGD) containing model peptide was conjugated to the surface of poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles as a ligand that can recognize adhesion molecules overexpressed on the surface of metastatic cancer cells, that is, integrins, and that can enhance the micellar delivery of encapsulated hydrophobic drug into a tumor cell. Toward this goal, PEO-b-PCL copolymers bearing acetal groups on the PEO end were synthesized, characterized, and assembled to polymeric micelles. The acetal group on the surface of the PEO-b-PCL micelles was converted to reactive aldehyde under acidic condition at room temperature. An RGD-containing linear peptide, GRGDS, was conjugated on the surface of the aldehyde-decorated PEO-b-PCL micelles by incubation at room temperature. A hydrophobic fluorescent probe, that is, DiI, was physically loaded in prepared polymeric micelles to imitate hydrophobic drugs loaded in micellar carrier. The cellular uptake of DiI loaded GRGDS-modified micelles by melanoma B16-F10 cells was investigated at 4 and 37 degrees C by fluorescent spectroscopy and confocal microscopy techniques and was compared to the uptake of DiI loaded valine-PEO-b-PCL micelles (as the irrelevant ligand decorated micelles) and free DiI. GRGDS conjugation to polymeric micelles significantly facilitated the cellular uptake of encapsulated hydrophobic DiI most probably by intergrin-mediated cell attachment and endocytosis. The results indicate that acetal-terminated PEO-b-PCL micelles are amenable for introducing targeting moieties on the surface of polymeric micelles and that RGD-peptide conjugated PEO-b-PCL micelles are promising ligand-targeted carriers for enhanced drug delivery to metastatic tumor cells.     

Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain

Conjugation to cationic cell penetrating peptides (such as Tat, Penetratin, or oligo arginines) efficiently improves the cellular uptake of large hydrophilic molecules such as oligonucleotides and peptide nucleic acids, but the cellular uptake is predominantly via an unproductive endosomal pathway and therefore mechanisms that promote endosomal escape (or avoid the endosomal route) are required for improving bioavailability. A variety of auxiliary agents (chloroquine, calcium ions, or lipophilic photosensitizers) has this effect, but improved, unaided delivery would be highly advantageous in particular for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases with increasing length of the fatty acid (C8-C16) but in parallel also results in increased cellular toxicity, with decanoic acid being optimal. Furthermore, the relative enhancement is significantly higher for Tat peptide compared to oligoarginine. Confocal microscopy and chloroquine enhancement indicates that the lipophilic domain increases the endosomal uptake as well as promoting significantly endosomal escape. These results provide a novel route for improving the (cellular) bioavailability of larger hydrophilic molecules.