Vectors For Intracellular Delivery Research Articles

C-di-GMP@ZIF-8 nanocomposite injectable hydrogel based on modified chitosan and hyaluronic acid for infected wound healing by activating STING signaling

The treatment of infected wounds relies on antibiotics; however, increasing drug resistance has made therapeutic processes more difficult. Activating self-innate immune abilities may provide a promising alternative to treat wounds with bacterial infections. In this work, we constructed an immunogenic injectable hydrogel crosslinked by the Schiff base reaction of carboxymethyl chitosan (NOCC) and aldehyde hyaluronic acid (AHA) and encapsulated with stimulator of interferon genes (STING) agonist c-di-GMP loaded ZIF-8 nanoparticles (c-di-GMP@ZIF-8). Nanocubic ZIF-8 was screened as the most efficient intracellular drug delivery vector from five differently-shaped morphologies. The NOCC/AHA hydrogel released c-di-GMP@ZIF-8 more quickly (43 %) in acidic environment (pH = 5.5) of infected wounds compared with 34 % in non-infected wound environment (pH = 7.4) at 96 h due to pH-responsive degradation performance. The released c-di-GMP@ZIF-8 was found to activate the STING signaling of macrophages and enhance the secretion of IFN-β, CCL2, and CXCL12 5.8–7.6 times compared with phosphate buffer saline control, which effectively inhibited S. aureus growth and promoted fibroblast migration. In rat models with infected wounds, the c-di-GMP@ZIF-8 nanocomposite hydrogels improved infected wound healing by promoting granulation tissue regeneration, alleviating S. aureus-induced inflammation, and improving angiogenesis. Altogether, this study demonstrated a feasible strategy using STING-targeted and pH-responsive hydrogels for infected wound management.

Functional L-Arginine Derivative as an Efficient Vector for Intracellular Protein Delivery for Potential Cancer Therapy.

The utilization of cytosolic protein delivery is a promising approach for treating various diseases by replacing dysfunctional proteins. Despite the development of various nanoparticle-based intracellular protein delivery methods, the complicated chemical synthesis of the vector, loading efficiency and endosomal escape efficiency of proteins remain a great challenge. Recently, 9-fluorenylmethyloxycarbonyl (Fmoc)-modified amino acid derivatives have been used to self-assemble into supramolecular nanomaterials for drug delivery. However, the instability of the Fmoc group in aqueous medium restricts its application. To address this issue, the Fmoc ligand neighboring arginine was substituted for dibenzocyclooctyne (DBCO) with a similar structure to Fmoc to obtain stable DBCO-functionalized L-arginine derivative (DR). Azide-modified triethylamine (crosslinker C) was combined with DR to construct self-assembled DRC via a click chemical reaction for delivering various proteins, such as BSA and saporin (SA), into the cytosol of cells. The hyaluronic-acid-coated DRC/SA was able to not only shield the cationic toxicity, but also enhance the intracellular delivery efficiency of proteins by targeting CD44 overexpression on the cell membrane. The DRC/SA/HA exhibited higher growth inhibition efficiency and lower IC50 compared to DRC/SA toward various cancer cell lines. In conclusion, DBCO-functionalized L-arginine derivative represents an excellent potential vector for protein-based cancer therapy.

Dual-Functionalized Covalent Triazine Framework Nanosheets as Hierarchical Nonviral Vectors for Intracellular Gene Delivery

Gene therapy is a revolutionary biomedical technique for the treatment of many diseases, and the development of safe and efficient intracellular gene delivery vectors is crucial for successful gene...

Modular Synthesis of Trifunctional Peptide-oligonucleotide Conjugates via Native Chemical Ligation.

Cell penetrating peptides (CPPs) are being increasingly used as efficient vectors for intracellular delivery of biologically active agents, such as therapeutic antisense oligonucleotides (ASOs). Unfortunately, ASOs have poor cell membrane permeability. The conjugation of ASOs to CPPs have been shown to significantly improve their cellular permeability and therapeutic efficacy. CPPs are often covalently conjugated to ASOs through a variety of chemical linkages. Most of the reported approaches for ligation of CPPs to ASOs relies on methodologies that forms non-native bond due to incompatibility with in-solution phase conjugation. These approaches have low efficiency and poor yields. Therefore, in this study, we have exploited native chemical ligation (NCL) as an efficient strategy for synthesizing CPP-ASO conjugates. A previously characterized CPP [ApoE(133–150)] was used to conjugate to a peptide nucleic acid (PNA) sequence targeting human survival motor neuron-2 (SMN2) mRNA which has been approved by the FDA for the treatment of spinal muscular atrophy. The synthesis of ApoE(133–150)-PNA conjugate using chemo-selective NCL was highly efficient and the conjugate was obtained in high yield. Toward synthesizing trifunctional CPP-ASO conjugates, we subsequently conjugated different functional moieties including a phosphorodiamidate morpholino oligonucleotide (PMO), an additional functional peptide or a fluorescent dye (Cy5) to the thiol that was generated after NCL. The in vitro analysis of the bifunctional CPP-PNA and trifunctional CPP-(PMO)-PNA, CPP-(peptide)-PNA and CPP-(Cy5)-PNA showed that all conjugates are cell-permeable and biologically active. Here we demonstrated chemo-selective NCL as a highly efficient and superior conjugation strategy to previously published methods for facile solution-phase synthesis of bi-/trifunctional CPP-ASO conjugates.

In silico identification and experimental validation of cellular uptake by a new cell penetrating peptide P1 derived from MARCKS

Viral vectors for vaccine delivery are challenged by recently reported safety issues like immunogenicity and risk for cancer development, and thus there is a growing need for the development of non-viral vectors. Cell penetrating peptides (CPPs) are non-viral vectors that can enter plasma membranes efficiently and deliver a broad range of cargoes. Our bioinformatic prediction and wet-lab validation data suggested that peptide P1 derived from MARCKS protein phosphorylation site domain is a new potential CPP candidate. We found that peptide P1 can efficiently internalize into various cell lines in a concentration-dependent manner. Receptor-mediated endocytosis pathway is the major mechanism of P1 penetration, although P1 also directly penetrates the plasma membrane. We also found that peptide P1 has low cytotoxicity in cultured cell lines as well as mouse red blood cells. Furthermore, peptide P1 not only can enter into cultured cells itself, but it also can interact with plasmid DNA and mediate the functional delivery of plasmid DNA into cultured cells, even in hard-to-transfect cells. Combined, these findings indicate that P1 may be a promising vector for efficient intracellular delivery of bioactive cargos.

Mesoporous additive-free vaterite CaCO3 crystals of untypical sizes: From submicron to Giant

Mesoporous CaCO3 crystals of sizes in the range 3–15 μm have attracted significant scientific attention as fully decomposable biocompatible drug delivery vectors. They are also used as sacrificial templates to produce polymer-based microcarriers (capsules, beads) and polymer scaffolds for tissue engineering at biologically friendly conditions. However, fabrication of CaCO3 crystals with the sizes beyond the conventional range without additives is still a challenge. There is a desperate need of sub-micrometre and sub-millimetre biocompatible and easily decomposable vectors for intracellular delivery and design of tailor-made polymer scaffolds, respectively. In this study, additive-free one-step fabrication of vaterite mesoporous crystals with sizes in the range from submicron (0.72 μm) up to tens of microns (55 μm) sizes is proposed. Crystals are grown by mixing of Ca2+ and CO32− salts in aqueous media. Internal structure and morphology of the crystals have been investigated. Encapsulation of lysozyme by means of co-synthesis shows an enhanced entrapment into submicron crystals. Besides, the approach to grow CaCO3 crystals on the surface of microcellulose ьш is demonstrated opening broad avenues for the development of vaterite coatings technologies.

Low molecular weight chitosan nanoparticles for CpG oligodeoxynucleotides delivery: Impact of molecular weight, degree of deacetylation, and mannosylation on intracellular uptake and cytokine induction

Although synthetic CpG oligodeoxynucleotides (ODNs) have shown substantial potential as immunotherapeutic agents, their effective intracellular delivery remains challenging. In this work, nanoparticles prepared from low-molecular weight (LMW) chitosans were investigated as CpG ODN delivery systems. Chitosan samples with a molecular weight (Mw) of 5 and 15 kDa and degree of deacetylation (DDA) of 50 and 80% were prepared. Additionally, mannosylated chitosans with a substitution degree of 15% were synthesized. The impact of LMW chitosan Mw and DDA on nanoparticle physical properties and the associated immunostimulatory effect in RAW 264.7 cells was studied. Nanoparticles prepared with chitosan of higher DDA and larger Mw exhibited better CpG ODN binding ability and intracellular uptake. Nevertheless, the most efficient immunostimulatory effect was observed while using 50% acetylated and mannosylated samples. The decreased charge density on chitosan backbone resulted in the enhanced intracellular CpG ODN release, which promoted in vitro cytokine secretion. Moreover, mannose ligand grafting promoted nanoparticle uptake through receptor-mediated recognition. Overall, this research suggests that chitosan structural parameters can be modulated to prepare LMW chitosan nanoparticles that first efficiently encapsulate CpG ODN, and then release it in immune cells, thus may be used as an efficient vector for intracellular CpG ODN delivery.

Acylation of the S413-PV cell-penetrating peptide as a means of enhancing its capacity to mediate nucleic acid delivery: Relevance of peptide/lipid interactions

BackgroundCell-penetrating peptides (CPPs) have been extensively exploited in gene therapy approaches as vectors for intracellular delivery of bioactive molecules. The ability of CPPs to be internalized into cells and their capacity to complex nucleic acids depend on their molecular structure, both primary and secondary, namely regarding hydrophobicity/hydrophilicity. CPP acylation has been used as a strategy to improve this structural feature. MethodsAcyl groups (from 6 to 18 carbon atoms) were attached to the S413-PV peptide and their effects on the peptide competence to complex siRNAs and to mediate gene silencing in glioblastoma (GBM) cells were studied. A systematic characterization of membrane interactions with S413-PV acyl-derivatives was also conducted, using different biophysical techniques (surface pressure-area isotherms in Langmuir monolayers, DSC and 31P NMR) to unravel a relationship between CPP biological activity and CPP effects on membrane stability and lipid organization. ResultsA remarkable concordance was noticed between acylated-S413-PV peptide competence to promote gene silencing in GBM cells and disturbance induced in membrane models, the lauroyl- and myristoyl-S413-PV peptides being the most effective. A cut-off effect was described for the first time regarding the influence of acyl-chain length on CPP bioactivity. ConclusionsC12-S413-PV showed high capacity to destabilize lipid bilayers, to escape from lysosomal degradation and to mediate gene silencing without promoting cytotoxicity. General significanceBesides unraveling a new CPP with high potential to be employed as a gene delivery vector, this work emphasizes the benefit from allying biophysical and biological studies towards a proper CPP structural refinement for successful pre-clinical/clinical application.

(Arg)9-SH2 superbinder: a novel promising anticancer therapy to melanoma by blocking phosphotyrosine signaling

BackgroundMelanoma is a malignant tumor with high misdiagnosis rate and poor prognosis. The bio-targeted therapy is a prevailing method in the treatment of melanoma; however, the accompanying drug resistance is inevitable. SH2 superbinder, a triple-mutant of the Src Homology 2 (SH2) domain, shows potent antitumor ability by replacing natural SH2-containing proteins and blocking multiple pY-based signaling pathways. Polyarginine (Arg)9, a powerful vector for intracellular delivery of large molecules, could transport therapeutic agents across cell membrane. The purpose of this study is to construct (Arg)9-SH2 superbinder and investigate its effects on melanoma cells, expecting to provide potential new approaches for anti-cancer therapy and overcoming the unavoidable drug resistance of single-targeted antitumor agents.Methods(Arg)9 and SH2 superbinder were fused to form (Arg)9-SH2 superbinder via genetic engineering. Pull down assay was performed to identify that (Arg)9-SH2 superbinder could capture a wide variety of pY proteins. Immunofluorescence was used to detect the efficiency of (Arg)9-SH2 superbinder entering cells. The proliferation ability was assessed by MTT and colony formation assay. In addition, wound healing and transwell assay were performed to evaluate migration of B16F10, A375 and A375/DDP cells. Moreover, apoptosis caused by (Arg)9-SH2 superbinder was analyzed by flow cytometry-based Annexin V/PI. Furthermore, western blot revealed that (Arg)9-SH2 superbinder influenced some pY-related signaling pathways. Finally, B16F10 xenograft model was established to confirm whether (Arg)9-SH2 superbinder could restrain the growth of tumor.ResultsOur data showed that (Arg)9-SH2 superbinder had the ability to enter melanoma cells effectively and displayed strong affinities for various pY proteins. Furthermore, (Arg)9-SH2 superbinder could repress proliferation, migration and induce apoptosis of melanoma cells by regulating PI3K/AKT, MAPK/ERK and JAK/STAT signaling pathways. Importantly, (Arg)9-SH2 superbinder could significantly inhibit the growth of tumor in mice.Conclusions(Arg)9-SH2 superbinder exhibited high affinities for pY proteins, which showed effective anticancer ability by replacing SH2-containing proteins and blocking diverse pY-based pathways. The remarkable ability of (Arg)9-SH2 superbinder to inhibit cancer cell proliferation and tumor growth might open the door to explore the SH2 superbinder as a therapeutic agent for cancer treatment.

Indoloazepinone-Constrained Oligomers as Cell-Penetrating and Blood-Brain-Barrier-Permeating Compounds.

Non-cationic and amphipathic indoloazepinone-constrained (Aia) oligomers have been synthesized as new vectors for intracellular delivery. The conformational preferences of the [l-Aia-Xxx]n oligomers were investigated by circular dichroism (CD) and NMR spectroscopy. Whereas Boc-[l-Aia-Gly]2,4 -OBn oligomers 12 and 13 and Boc-[l-Aia-β3 -h-l-Ala]2,4 -OBn oligomers 16 and 17 were totally or partially disordered, Boc-[l-Aia-l-Ala]2 -OBn (14) induced a typical turn stabilized by C5 - and C7 -membered H-bond pseudo-cycles and aromatic interactions. Boc-[l-Aia-l-Ala]4 -OBn (15) exhibited a unique structure with remarkable T-shaped π-stacking interactions involving the indole rings of the four l-Aia residues forming a dense hydrophobic cluster. All of the proposed FITC-6-Ahx-[l-Aia-Xxx]4 -NH2 oligomers 19-23, with the exception of FITC-6-Ahx-[l-Aia-Gly]4 -NH2 (18), were internalized by MDA-MB-231 cells with higher efficiency than the positive references penetratin and Arg8 . In parallel, the compounds of this series were successfully explored in an in vitro blood-brain barrier (BBB) permeation assay. Although no passive diffusion permeability was observed for any of the tested Ac-[l-Aia-Xxx]4 -NH2 oligomers in the PAMPA model, Ac-[l-Aia-l-Arg]4 -NH2 (26) showed significant permeation in the in vitro cell-based human model of the BBB, suggesting an active mechanism of cell penetration.

Degradation Behavior of Silk Nanoparticles-Enzyme Responsiveness.

Silk nanoparticles are viewed as promising vectors for intracellular drug delivery as they can be taken up into cells by endocytosis and trafficked to lysosomes, where lysosomal enzymes and the low pH trigger payload release. However, the subsequent degradation of the silk nanoparticles themselves still requires study. Here, we report the responsiveness of native and PEGylated silk nanoparticles to degradation following exposure to proteolytic enzymes (protease XIV and α-chymotrypsin) and papain, a cysteine protease. Both native and PEGylated silk nanoparticles showed similar degradation behavior over a 20 day exposure period (degradation rate: protease XIV > papain ≫ α-chymotrypsin). Within 1 day, the silk nanoparticles were rapidly degraded by protease XIV, resulting in a ∼50% mass loss, an increase in particle size, and a reduction in the amorphous content of the silk secondary structure. By contrast, 10 days of papain treatment was necessary to observe any significant change in nanoparticle properties, and α-chymotrypsin treatment had no effect on silk nanoparticle characteristics over the 20-day study period. Silk nanoparticles were also exposed ex vivo to mammalian lysosomal enzyme preparations to mimic the complex lysosomal microenvironment. Preliminary results indicated a 45% reduction in the silk nanoparticle size over a 5-day exposure. Overall, the results demonstrate that silk nanoparticles undergo enzymatic degradation, but the extent and kinetics are enzyme-specific.

Polycation-telodendrimer nanocomplexes for intracellular protein delivery

Intracellular delivery of protein therapeutics by cationic polymer vehicles is an emerging technique that is, however, encountering poor stability, high cytotoxicity and non-specific cell uptake. Herein, we present a facile strategy to optimize the protein-polycation complexes by encapsulating with linear-dendritic telodendrimers. The telodendrimers with well-defined structures enable the rational design and integration of multiple functionalities for efficient encapsulation of the protein-polycation complexes by multivalent and hybrid supramolecular interactions to produce sub-20 nm nanoparticles. This strategy not only reduces the polycation-associated cytotoxicity and hemolytic activity, but also eliminates the aggregation and non-specific binding of polycations to other biomacromolecules. Moreover, the telodendrimers dissociate readily from the complexes during the cellular uptake process, which restores the capability of polycations for intracellular protein delivery. This strategy overcomes the limitations of polycationic vectors for intracellular delivery of protein therapeutics.

Intracellular and transdermal protein delivery mediated by non-covalent interactions with a synthetic guanidine-rich molecular carrier

The impermeability of the cell plasma membrane is one of the major barriers for protein transduction into mammalian cells, and it also limits the use of proteins as therapeutic agents. Protein transduction has usually been achieved based on certain invasive processes or cell penetrating peptides (CPP). Herein we report our study in which a synthetic guanidine-rich molecular carrier is used as a delivery vector for intracellular and transdermal delivery of proteins. First a sorbitol-based molecular carrier having 8 guanidine units (Sor-G8) was synthesized, and then was simply mixed with a cargo protein of varying sizes to form the non-covalent complex of carrier-cargo proteins. These ionic complexes were shown to have efficient cellular uptake properties. The optimum conditions including the molar ratio between cargo protein and carrier, and the treatment time have been defined. Several protein cargoes were successfully examined with differing sizes and molecular weights: green fluorescent protein (MW 27kDa), albumin (66kDa), concanavalin A (102kDa), and immunoglobulin G (150kDa). These non-covalent complexes were also found to have excellent transdermal penetration ability into the mouse skin. The skin penetration depth was studied histologically by light microscopy as well as two-photon microscopy thus generating a depth profile. These complexes were largely found in the epidermis and dermis layers, i.e. down to ca. 100μm depth of the mouse skin. Our synthetic Sor-G8 carrier was found to be substantially more efficient that Arg8 in both the intracellular transduction and the transdermal delivery of proteins. The mechanism of the cellular uptake of the complex was briefly studied, and the results suggested macropinocytosis.

Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells

AbstractDespite extensive use of arginine‐rich cell‐penetrating peptides (CPPs)—including octaarginine (R8)—as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity‐sensitive dye (di‐4‐ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature‐inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.

Loosening of Lipid Packing Promotes Oligoarginine Entry into Cells.

Despite extensive use of arginine-rich cell-penetrating peptides (CPPs)-including octaarginine (R8)-as intracellular delivery vectors, mechanisms for their internalization are still under debate. Lipid packing in live cell membranes was characterized using a polarity-sensitive dye (di-4-ANEPPDHQ), and evaluated in terms of generalized polarization. Treatment with membrane curvature-inducing peptides led to significant loosening of the lipid packing, resulting in an enhanced R8 penetration. Pyrenebutyrate (PyB) is known to facilitate R8 membrane translocation by working as a hydrophobic counteranion. Interestingly, PyB also actively induced membrane curvature and perturbed lipid packing. R8 is known to directly cross cell membranes at elevated concentrations. The sites of R8 influx were found to have looser lipid packing than surrounding areas. Lipid packing loosening is proposed as a key factor that governs the membrane translocation of CPPs.

Tetrahedral DNA Nanoparticle Vector for Intracellular Delivery of Targeted Peptide Nucleic Acid Antisense Agents to Restore Antibiotic Sensitivity in Cefotaxime-Resistant Escherichia coli.

The bacterial cell wall presents a barrier to the uptake of unmodified synthetic antisense oligonucleotides, such as peptide nucleic acids, and so is one of the greatest obstacles to the development of their use as therapeutic anti-bacterial agents. Cell-penetrating peptides have been covalently attached to antisense agents, to facilitate penetration of the bacterial cell wall and deliver their cargo into the cytoplasm. Although they are an effective vector for antisense oligonucleotides, they are not specific for bacterial cells and can exhibit growth inhibitory properties at higher doses. Using a bacterial cell growth assay in the presence of cefotaxime (CTX 16 mg/L), we have developed and evaluated a self-assembling non-toxic DNA tetrahedron nanoparticle vector incorporating a targeted anti-blaCTX-M-group 1 antisense peptide nucleic acid (PNA4) in its structure for penetration of the bacterial cell wall. A dose-dependent CTX potentiating effect was observed when PNA4 (0-40 μM) was incorporated into the structure of a DNA tetrahedron vector. The minimum inhibitory concentration (to CTX) of an Escherichia coli field isolate harboring a plasmid carrying blaCTX-M-3 was reduced from 35 to 16 mg/L in the presence of PNA4 carried by the DNA tetrahedron vector (40 μM), contrasting with no reduction in MIC in the presence of PNA4 alone. No growth inhibitory effects of the DNA tetrahedron vector alone were observed.

Intracellular plasmid DNA delivery by self-assembled nanoparticles of amphiphilic PHML-b-PLLA-b-PHML copolymers and the endocytosis pathway analysis.

This work presents a new series of polycationic nanoparticles of (l-)-lysine conjugated amphiphilic triblock copolymer poly(hydroxyletheyl methacrylate-L-lysine)-b-poly(L-lactide)-b-poly(hydroxyletheyl methacrylate-L-lysine)s (PHML-b-PLLA-b-PHML) as potent low cytotoxic vectors for intracellular plasmid DNA delivery. First, the triblock PHML-b-PLLA-b-PHML copolymers were prepared via a combination of metal-free controlled ring opening polymerization and successive atom transfer radical polymerization. Then the cationic PHML-b-PLLA-b-PHML nanoparticles were further prepared by solution self-assembly. The particle size, zeta potential and morphology of as-prepared PHML-b-PLLA-b-PHML nanoparticles were characterized by dynamic light scattering and atomic force microscopy, respectively. The plasmid DNA binding affinities and polyplex stabilities were separately explored by agarose gel retardation and DNase I degradation assays. Then invitro cytotoxicity and gene transfection efficiency of the PHML-b-PLLA-b-PHML nanoparticles vectors as well as relevant polyplex endocytosis pathway were investigated with H1299 cells. It was revealed that the PHML-b-PLLA-b-PHML nanoparticles exhibited low cytotoxicity, strong plasmid DNA binding affinity, high polyplex stability and efficient plasmid DNA transfection even under serum conditions (10% FBS). Moreover, the endocytosis analysis results disclosed that the PHML30-b-PLLA-b-PHML30 nanoparticle/plasmid DNA polyplexes were predominantly involved in lipid-raft-mediated endocytosis pathway, similar to that of SV40 virus-based vectors.

Synthesis and application of poly(ethylene glycol)-co-poly(β-amino ester) copolymers for small cell lung cancer gene therapy

The design of polymeric nanoparticles for gene therapy requires engineering of polymer structure to overcome multiple barriers, including prolonged colloidal stability during formulation and application. Poly(β-amino ester)s (PBAEs) have been shown effective as polymeric vectors for intracellular DNA delivery, but limited studies have focused on polymer modifications to enhance the stability of PBAE/DNA polyplexes. We developed block copolymers consisting of PBAE oligomer center units and poly(ethylene glycol) (PEG) end units. We fabricated a library of PEG-PBAE polyplexes by blending PEGylated PBAEs of different PEG molecular weights and non-PEGylated PBAEs of different structures at various mass ratios of cationic polymer to anionic DNA. Non-PEGylated PBAE polyplexes aggregated following a 24h incubation in acidic and physiological buffers, presenting a challenge for therapeutic use. In contrast, among 36 PEG-PBAE polyplex formulations evaluated, certain polyplexes maintained a small size under these conditions. These selected polyplexes were further evaluated for transfection in human small cell lung cancer cells (H446) in the presence of serum, and the best formulation transfected ∼40% of these hard-to-transfect cells while preventing polymer-mediated cytotoxicity. When PEG-PBAE polyplex delivered Herpes simplex virus thymidine kinase plasmid in combination with the prodrug ganciclovir, the polyplexes killed significantly more H446 cancer cells (35%) compared to healthy human lung fibroblasts (IMR-90) (15%). These findings indicate that PEG-PBAE polyplexes can maintain particle stability without compromising their therapeutic function for intracellular delivery to human small cell lung cancer cells, demonstrate potential cancer specificity, and have potential as safe materials for small cell lung cancer gene therapy. Statement of SignificanceMany natural and synthetic biomaterials have been investigated as non-viral vectors to deliver nucleic acids for cancer therapy. However, there are multiple hurdles to successful transfection including achieving particle stability, efficient delivery to cancer cells, and low cytotoxicity. In particular, engineering the physicochemical surface properties of a nanoparticle to improve stability is often offset by a decrease in the cellular entry and transfection efficiency. We developed stable polymeric nanoparticles that demonstrate high transfection efficiency by modifying synthetic biodegradable cationic polymers and engineering nanoparticle formulations using a combinatorial approach. The results of this study show the potential of biodegradable surface-modified polymeric nanoparticles as clinically translatable, biomaterial-based vehicles for cancer gene therapy.

Abstract 5326: Intracellular bacterial delivery of a NIPP1-peptide kills tumor cells

Abstract Protein phosphatase-1 (PP1) is a cancer target because it is involved in many cell signaling pathways. Its activity is tightly regulated by its interaction partners, mainly via the RVxF-binding motif. RVxF-containing peptides deregulate PP1 and have therapeutic potential as antagonists of increased cancer kinase signaling. Because PP1 is responsible for more than half of the dephosphorylations on Ser/Thr sites, it is essential in all eukaryotic cells and it is difficult to target systemically. Moreover, delivering peptides to cells extracellularly is inefficient because of poor cell permeability and proteolytic instability. To specifically transport therapeutic peptides into cancer cells, we engineered bacteria to be intracellular delivery vectors. A bacterial delivery system (BDS) was designed by creating a strain that lyses after cell invasion and releases its contents into the cytoplasm of cancer cells. A BDS strain was then manipulated to express a peptide containing the RVxF motif of Nuclear Inhibitor of PP1 (NIPP1; AA 142-224). The therapeutic potential of this strain was tested on MCF-7 cell monolayers and on 3D tumor tissue. The BDS was cloned by inserting the bacterial phage lysis gene E under the control of the sseJ promoter, a Salmonella promoter that is activated after cell invasion. The specificity of the sseJ promoter was tested by inserting GFP under its control and infecting MCF-7 cancer cells with Salmonella containing the plasmid. All intracellular bacteria expressed GPF after 4 hours of infection, while no fluorescence was measured in the bacteria outside the mammalian cells. After infection, the created BDS lysed in 78% of invaded cells. BDS delivery of the NIPP1-peptide to MCF-7 cancer cells killed 41% of infected cells while infection with control, empty BDS did not result in any death. When treating tumor tissue with BDS expressing NIPP1-peptide, 39% of tumor tissue was killed after 9.5 hours, compared to 8% when treated with BDS alone. These results indicate that targeting phosphatases with intracellular peptide delivery by bacteria is feasible and reduces tumor tissue viability. Citation Format: Nele Van Dessel, Neil S. Forbes. Intracellular bacterial delivery of a NIPP1-peptide kills tumor cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5326. doi:10.1158/1538-7445.AM2015-5326

Efficient Delivery of Structurally Diverse Protein Cargo into Mammalian Cells by a Bacterial Toxin.

Platforms enabling targeted delivery of proteins into cells are needed to fully realize the potential of protein-based therapeutics with intracellular sites-of-action. Bacterial toxins are attractive systems to consider as templates for designing protein transduction systems as they naturally bind and enter specific cells with high efficiency. Here we investigated the capacity of diphtheria toxin to function as an intracellular protein delivery vector. We report that diphtheria toxin delivers an impressive array of passenger proteins spanning a range of sizes, structures, and stabilities into cells in a manner that indicates that they are "invisible" to the translocation machinery. Further, we show that α-amylase delivered into cells by a detoxified diphtheria toxin chimera digests intracellular glycogen in live cells, providing evidence that delivered cargo is folded, active, and abundant. The efficiency and versatility of diphtheria toxin over existing systems open numerous possibilities for intracellular delivery of bioactive proteins.