A review on gold nanoparticles as an innovative therapeutic cue in bone tissue engineering: Prospects and future clinical applications.

Bone damage is a complex orthopedic problem primarily caused by trauma, cancer, or bacterial infection of bone tissue. Clinical care management for bone damage remains a significant clinical challenge and there is a growing need for more advanced bone therapy options. Nanotechnology has been widely explored in the field of orthopedic therapy for the treatment of a severe bone disease. Among nanomaterials, gold nanoparticles (GNPs) along with other biomaterials are emerging as a new paradigm for treatment with excellent potential for bone tissue engineering and regenerative medicine applications. In recent years, a great deal of research has focused on demonstrating the potential for GNPs to provide for enhancement of osteogenesis, reduction of osteoclastogenesis/osteomyelitis, and treatment of bone cancer. This review details the latest understandings in regards to GNPs based therapeutic systems, mechanisms, and the applications of GNPs against various bone disorders. The present review aims to summarize i) the mechanisms of GNPs in bone tissue remodeling, ii) preparation methods of GNPs, and iii) functionalization of GNPs and its decoration on biomaterials as a delivery vehicle in a specific bone tissue engineering for future clinical application.

Lipid core-shell nanoparticles co-deliver FOLFOX regimen and siPD-L1 for synergistic targeted cancer treatment.

FOLFOX regimen, composed of folinic acid, 5-fluorouracil (5-FU) and oxaliplatin (OXP), has been used as clinical standard therapeutic regimen in treatments of colorectal cancer (CRC) and esophageal squamous cell carcinoma (ESCC). To further improve its therapeutic outcomes, FOLFOX was combined with anti-PD-1 antibody to form an advanced chemo-immune combination strategy, which has been proven more efficient in controlling cancer progression and prolonging patients' survival in various clinical trials. However, bad tumor accumulation, relative high toxicity, numerous treatment cycles with high fees and low compliance as well as drug resistance seriously limit the prognosis of FOLFOX regimen. The "all-in-one" formulations, which could precisely delivery multidrug regimen into tumor sites and cells, showed a promising application prospect for targeted drug delivery as well as reducing side effects. However, the design and preparation of the "all-in-one" formulation with high drug encapsulation efficiencies for all drugs was still challenging. Herein, a lipid core-shell nanoparticle codelivery platform was designed for simultaneous encapsulation of variant FOLFOX composed of miriplatin (MiPt), 5-Fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP), calcium folinate (CF) and PD-L1 siRNA (siPD-L1) with high efficiencies, and their synergistic anti-tumor mechanisms were studied, respectively. MiPt, a precursor of OXP, was validated capable of inducing efficient immunogenic cell death (ICD) in this work. Additionally, ICD-mediated release of damage associated molecular patterns functionalized synergistically with PD-L1 silence by siPD-L1 to overcome chemoresistance, reverse suppressive tumor microenvironment and recruit more CD8+ T cells. FdUMP, as the intracellular active form of 5-FU, could induce large amounts of reactive oxygen species to enhance the ICD. CF worked as the sensitizer of FdUMP. The enhanced long-term anti-tumor effect of the prepared "all-in-one" formulation compared to free drug regimen and other controls, was verified in heterotopic CRC mice models and ESCC mice models, providing new thoughts for researchers and showing a promising prospect of translation into clinical applications.

An injectable signal-amplifying device elicits a specific immune response against malignant glioblastoma.

Despite exciting achievements with some malignancies, immunotherapy for hypoimmunogenic cancers, especially glioblastoma (GBM), remains a formidable clinical challenge. Poor immunogenicity and deficient immune infiltrates are two major limitations to an effective cancer-specific immune response. Herein, we propose that an injectable signal-amplifying nanocomposite/hydrogel system consisting of granulocyte-macrophage colony-stimulating factor and imiquimod-loaded antigen-capturing nanoparticles can simultaneously amplify the chemotactic signal of antigen-presenting cells and the "danger" signal of GBM. We demonstrated the feasibility of this strategy in two scenarios of GBM. In the first scenario, we showed that this simultaneous amplification system, in conjunction with local chemotherapy, enhanced both the immunogenicity and immune infiltrates in a recurrent GBM model; thus, ultimately making a cold GBM hot and suppressing postoperative relapse. Encouraged by excellent efficacy, we further exploited this signal-amplifying system to improve the efficiency of vaccine lysate in the treatment of refractory multiple GBM, a disease with limited clinical treatment options. In general, this biomaterial-based immune signal amplification system represents a unique approach to restore GBM-specific immunity and may provide a beneficial preliminary treatment for other clinically refractory malignancies.

Antitumor synergism between PAK4 silencing and immunogenic phototherapy of engineered extracellular vesicles.

Immunotherapy has revolutionized the landscape of cancer treatment. However, single immunotherapy only works well in a small subset of patients. Combined immunotherapy with antitumor synergism holds considerable potential to boost the therapeutic outcome. Nevertheless, the synergistic, additive or antagonistic antitumor effects of combined immunotherapies have been rarely explored. Herein, we established a novel combined cancer treatment modality by synergizing p21-activated kinase 4 (PAK4) silencing with immunogenic phototherapy in engineered extracellular vesicles (EVs) that were fabricated by coating M1 macrophage-derived EVs on the surface of the nano-complex cores assembled with siRNA against PAK4 and a photoactivatable polyethyleneimine. The engineered EVs induced potent PAK4 silencing and robust immunogenic phototherapy, thus contributing to effective antitumor effects in vitro and in vivo. Moreover, the antitumor synergism of the combined treatment was quantitatively determined by the CompuSyn method. The combination index (CI) and isobologram results confirmed that there was an antitumor synergism for the combined treatment. Furthermore, the dose reduction index (DRI) showed favorable dose reduction, revealing lower toxicity and higher biocompatibility of the engineered EVs. Collectively, the study presents a synergistically potentiated cancer treatment modality by combining PAK4 silencing with immunogenic phototherapy in engineered EVs, which is promising for boosting the therapeutic outcome of cancer immunotherapy.

Dual-sensitive drug-loaded hydrogel system for local inhibition of post-surgical glioma recurrence.

Local treatment after resection to inhibit glioma recurrence is thought to able to meet the real medical needs. However, the only clinically approved local glioma treatment-wafer containing bis(2-chloroethyl) nitrosourea (BCNU) showed very limited effects. Herein, in order to inhibit tumor recurrence with prolonged and synergistic therapeutic effect of drugs after tumor resection, an in situ dual-sensitive hydrogel drug delivery system loaded with two synergistic chemo-drugs BCNU and temozolomide (TMZ) was developed. The thermosensitive hydrogel was loaded with reactive oxygen species (ROS)-sensitive poly (lactic-co-glycolic) acid nanoparticles (NPs) encapsulating both BCNU and TMZ and also free BCNU and TMZ. The in vitro synergistic effect of BCNU and TMZ and in vivo presence of ROS at the residual tumor site were confirmed. The prepared ROS-sensitive NPs and thermosensitive hydrogel, as well as the long-term release behavior of drugs and NPs, were fully characterized both in vitro and in vivo. After >90% glioblastoma resection, the dual-sensitive hydrogel drug delivery system was injected into the resection cavity. The median survival time of the experimental group reached 65 days which was twice as long as the Resection only group, implying that this in situ drug delivery system effectively inhibited tumor recurrence. Overall, this study provides new ideas and strategies for the inhibition of postoperative glioma recurrence.

Antibody-siRNA conjugates (ARCs) using multifunctional peptide as a tumor enzyme cleavable linker mediated effective intracellular delivery of siRNA.

The tissue-specific targeted delivery and efficient cellular uptake of siRNAs are the main obstacles to their clinical application. Antibody-siRNA-conjugates (ARCs) can deliver siRNA by exploiting the targeting property of antibodies like antibody-drug conjugates (ADCs). However, the effective conjugation of antibodies and siRNAs and the release of siRNAs specifically at target sites have posed challenges to the development of ARCs. In this study, the successful conjugation of antibodies and siRNAs was achieved using a multifunctional peptide as a linker, composed of a cell-penetrating peptide (CPP) and a substrate peptide (SP), which is highly expressed in solid tumors. The resulting antibody-multifunctional peptide (SP-CPP)-siRNA system delivered the siRNA to target tumor cells by the specific binding of the antibody. Once the enzymes on the tumor cell surface hydrolyzed the substrate peptide linker, siRNA-CPP was released from ARCs. The released siRNA-CPP entered the targeted cells via the cellular penetrating ability of CPP, resulting in improved siRNA-mediated gene silencing efficiency, verified both in vitro and in vivo. After intravenous administration, the designed ARCs achieved approximately 66.7% EGFP (Enhanced Green Fluorescent Protein) downregulation efficiency in nude mice xenografted with the HCT116-EGFP tumor model. The proposed system provides a prospective choice for ARC production and the safe and efficient delivery of siRNAs.

Cell-penetrating peptide enhanced insulin buccal absorption.

Non-injectable delivery of peptides and proteins is not feasible due to the limitations of large molecular mass, high hydrophilic properties, and gastrointestinal degradation. Therefore, proposing a new method to solve this problem is a burning issue. The objective of this study was to propose a novel protein delivery strategy to overcome the poor efficacy and irritation of buccal insulin delivery. In this study, we applied a conjugate of cell-penetrating peptides (LMWP) and insulin (INS-PEG-LMWP) for buccal delivery. INS-PEG-LMWP was prepared using insulin solution and mixture as references. The transport behaviour, in vivo bioactivity, hypoglycaemic effect, and safety of INS-PEG-LMWP were systematically characterised. An in vitro study demonstrated that the uptake and transportation of INS-PEG-LMWP across buccal mucosal multilayers significantly increased. By comparing the effects of different endocytic inhibitors on INS-PEG-LMWP uptake, the conjugate might be delivered via an energy independent, electrostatically adsorbed pathway. INS-PEG-LMWP's relative pharmacological bioavailability was high and its relative bioavailability was up to 26.86%, demonstrating no visible mucosal irritation. Cell-penetrating peptides are likely to become a reliable and safe tool for overcoming insulin's low permeability through the epithelial multilayers, the major barrier to buccal delivery.

Local delivery of cardiac stem cells overexpressing HIF-1α promotes angiogenesis and muscular tissue repair in a hind limb ischemia model.

Critical limb ischemia (CLI) is the most advanced stage of peripheral artery disease, associated with significant risk of limb loss, morbidity and mortality; however, there remains unmet therapeutic needs for arterial revascularization and ischemic tissue repair. Stem cell therapies have emerged as compelling candidates due to beneficial proangiogenic and immunosuppressive function. Nevertheless, in vivo efficacy was insufficient in proliferation, differentiation and survival/engraftment rate. Cardiac stem cells (CSCs) was firstly attempted for CLI as a novel therapeutic modality to provide superior angiogenic potency to bone marrow-derived stem cells (BMSCs). It was noted that CSCs demonstrated 3.2-fold in HGF, 2.9-fold in VEGF and 8.7-fold in PDGF-B higher gene expressions compared to BMSCs. To enhance the hypoxia-induced proangiogenic effect, CSCs were transfected with hypoxia-inducible factor-1 alpha (HIF-1α) by using electroporation method, specifically optimized for CSCs yielding 45.77% of transfection efficiency and 89.75% of viability. HIF-1α overexpression significantly increased CSC survival in hypoxia, proangiogenic factors production and endothelial differentiation. In mouse hind limb ischemia model, local intramuscular delivery of CSC overexpressing HIF-1α (HIF-CSC) significantly improved the blood flow recovery. Histological analysis revealed that muscle degeneration and fibrosis in the ischemic limb were attenuated. Local delivery of HIF-CSC might be a promising option for ischemic tissue restoration.

MT1-MMP activatable fluorogenic probes with enhanced specificity via high-affinity peptide conjugation for tumor imaging.

Overlapping substrate specificities within the family of matrix metalloproteinases (MMPs), usually caused by their highly conserved structural topology, increase the potential for a substrate to be cleaved by multiple enzymes within this family, which leads to the decrease in the selectivity of MMP substrate-based probes. To resolve this issue, MT1-MMP activatable fluorogenic probes for tumor detection with enhanced specificity were developed by combining a fluorescence resonance energy transfer (FRET) peptide substrate and its specific binding peptide with different lengths of linkers. The specificity of the probes increased profiting from the high affinity of the MT1-MMP specific binding peptide while keeping the ability to amplify the output imaging signals in response to MMP activity with the FRET substrate. Enzyme kinetics analysis clearly demonstrated that the conjugation of P-1 and MT1-AF7p enhanced both the specificity and selectivity of the fluorogenic probes for MT1-MMP, and introducing a linker composed of 12 PEG subunits into these two fragments led to optimized specificity and selectivity of the fluorogenic probe for MT1-MMP. Both in vitro and in vivo results revealed that the imaging probe with the linker composed of 12 PEG subunits based on our designed strategy could be effectively applied for MT1-MMP positive tumor imaging. Since this strategy for enhancing the specificity of protease sensing probes can be applied to other proteases and is not just limited to MT1-MMP, it is an appealing platform to achieve selective tumor imaging.

A cis-Diol/pH Dual-Responsive Upconversion Nanoplatform: Synthesis, Characterization, and In Vitro Demonstration.

Integrating the functions of bioimaging, targeting and controlled release of therapeutic agents into a single nanoparticle is of great interests in nanomedicine and nanobiology. Herein, a cis -diol/pH dual-responsive upconversion nanoparticle (UCNP)-based theranostic platform has been developed for delivery of the anticancer drug to cancer cells. This nanoplatform is based on the strategic design of targetable hyaluronan modified UCNPs (HA-UCNPs) that are coupled with aminobenzeneboronic acid (APBA) to obtain APBA-UCNPs, having favorable tumor selectivity as well as the capacity for capturing cis-diol-containing therapeutics. The controlled release function is then achieved through the self-assembly of hydroxycamptothecin derivative ligands onto the surfaces of APBA-UCNPs, which is controllable in a stimuli-dependent manner. The UCNP-based theranostic probe taken up by tumor cells via receptor-mediated endocytosis liberates drugs triggered by competitive glucose at low pH in endosomes/lysosomes, resulting in cell apoptosis. The dual-responsive mechanism of boronate ester bonds gives a chemoselective strategy for controlled release of drug within tumor cells, establishing an alternative approach to treat a broad spectrum of diseases exploiting similar boronic acid-involved therapeutics.

Intra-articular delivery of synovium-resident mesenchymal stem cells via BMP-7-loaded fibrous PLGA scaffolds for cartilage repair.

Delivery of synovium-resident mesenchymal stem cells (synMSCs) to cartilage defect site might provide a novel therapeutic modality for treatment of articular cartilage diseases. However, low isolation efficiency of synMSCs limits their therapeutic application. Niche-preserving non-enzymatic isolation of synMSCs was firstly attempted by employing micro-organ culture system based on recapitulating tissue-specific homeostasis ex vivo. The isolated synMSCs retained superior long-term growth competency, proliferation and chondrogenic potential to bone marrow-derived MSCs (BMSCs). It was noted that synMSCs demonstrated 9-fold increase in cartilaginous micro-tissue formation and 13-fold increase in sulfated proteoglycans deposition compared to BMSCs. For delivery of synMSCs, fibrous PLGA scaffolds were specifically designed for full-thickness osteochondral defects in rabbits. The scaffolds provided effective micro-environment for growth and host-integration of synMSCs. Combined delivery of synMSCs with bone morphogenetic proteins-7 (BMP-7) was designed to achieve synergistic therapeutic efficacy. BMP-7-loaded PLGA nanoparticles electrosprayed onto the scaffolds released BMP-7 over 2 weeks to conform with its aimed role in stimulating early stage endochondral ossification. Scaffold-supported combined administration of synMSCs with BMP-7 resulted in high proteoglycan and collagen type II induction and thick hyaline cartilage formation. Intra-articular co-delivery of synMSCs with BMP-7 via fibrous PLGA scaffolds may be a promising therapeutic modality for articular cartilage repair.

Tat-functionalized Ag-Fe3O4 nano-composites as tissue-penetrating vehicles for tumor magnetic targeting and drug delivery.

In this paper, we prepared a dual functional system based on dextrin-coated silver nanoparticles which were further attached with iron oxide nanoparticles and cell penetrating peptide (Tat), producing Tat-modified Ag-Fe3O4 nanocomposites (Tat-FeAgNPs). To load drugs, an -SH containing linker, 3-mercaptopropanohydrazide, was designed and synthesized. It enabled the silver carriers to load and release doxorubicin (Dox) in a pH-sensitive pattern. The delivery efficiency of this system was assessed in vitro using MCF-7 cells, and in vivo using null BalB/c mice bearing MCF-7 xenograft tumors. Our results demonstrated that both Tat and externally applied magnetic field could promote cellular uptake and consequently the cytotoxicity of doxorubicin-loaded nanoparticles, with the IC50 of Tat-FeAgNP-Dox to be 0.63 µmol/L. The in vivo delivery efficiency of Tat-FeAgNP carrying Cy5 to the mouse tumor was analyzed using the in vivo optical imaging tests, in which Tat-FeAgNP-Cy5 yielded the most efficient accumulation in the tumor (6.7±2.4% ID of Tat-FeAgNPs). Anti-tumor assessment also demonstrated that Tat-FeAgNP-Dox displayed the most significant tumor-inhibiting effects and reduced the specific growth rate of tumor by 29.6% (P = 0.009), which could be attributed to its superior performance in tumor drug delivery in comparison with the control nanovehicles.

MMP-2-responsive fluorescent nanoprobes for enhanced selectivity of tumor cell uptake and imaging.

It is difficult to develop highly selective substrate-based fluorescent nanoprobes for specific matrix metalloproteinases (MMPs) due to overlapping substrate specificities among the family of MMP enzymes. To resolve this issue, we have developed novel fluorescent nanoprobes that are highly selective for soluble MMP-2. Herein, MMP-2-responsive nanoprobes were prepared by immobilizing fluorescent fusion proteins on nickel ferrite nanoparticles via the His-tag nickel chelation mechanism. The fusion protein consisted of a fluorescent mCherry protein with a cell penetrating peptide (CPP) moiety. An MMP-2 cleavage site was also introduced within the fusion protein, which was directly linked to the nickel ferrite nanoparticles. The selectivity of nanoprobes was modulated by hiding the cleavage site of MMP-2 substrates deeply inside the system, which could result in strong steric hindrance between the nanoprobes and MMPs, especially for membrane-tethered MMPs such as MMP-14. A cell-based assay demonstrated that the nanoprobes could only be activated by tumor cells secreting soluble MMP-2, but not membrane-tethered MMP-14. To further evaluate the contribution of the steric hindrance effect on the nanoprobes, a truncated recombinant MMP-14 was employed to confer their cleavage activity as compared to native membrane-tethered MMP-14. Furthermore, a designed probe with a diminished steric hindrance effect was proved to be activated by membrane-tethered type MMP-14. The results indicated that the design of fluorescent nanoprobes employing the steric hindrance effect can greatly enhance the selectivity of MMP-responsive nanoprobes realizing the specific detection of soluble MMP-2 in a tumor microenvironment. We believe that highly selective MMP-2-responsive fluorescent nanoprobes have broad impacts on biomedical applications including molecular imaging and labeling for tumor detection.

Sustained Release of Immunosuppressant by Nanoparticle-anchoring Hydrogel Scaffold Improved the Survival of Transplanted Stem Cells and Tissue Regeneration.

The outcome of scaffold-based stem cell transplantation remains unsatisfied due to the poor survival of transplanted cells. One of the major hurdles associated with the stem cell survival is the immune rejection, which can be effectively reduced by the use of immunosuppressant. However, ideal localized and sustained release of immunosuppressant is difficult to be realized, because it is arduous to hold the drug delivery system within scaffold for a long period of time. In the present study, the sustained release of immunosuppressant for the purpose of improving the survival of stem cells was successfully realized by a nanoparticle-anchoring hydrogel scaffold we developed. Methods: Poly (lactic-co-glycolic acid) (PLGA) nanoparticles were modified with RADA16 (RNPs), a self-assembling peptide, and then anchored to a RADA16 hydrogel (RNPs + Gel). The immobilization of RNPs in hydrogel was measured in vitro and in vivo, including the Brownian motion and cumulative leakage of RNPs and the in vivo retention of injected RNPs with hydrogel. Tacrolimus, as a typical immunosuppressant, was encapsulated in RNPs (T-RNPs) that were anchored to the hydrogel and its release behavior were studied. Endothelial progenitor cells (EPCs), as model stem cells, were cultured in the T-RNPs-anchoring hydrogel to test the immune-suppressing effect. The cytotoxicity of the scaffold against EPCs was also measured compared with free tacrolimus-loaded hydrogel. The therapeutic efficacy of the scaffold laden with EPCs on the hind limb ischemia was further evaluated in mice. Results: The Brownian motion and cumulative leakage of RNPs were significantly decreased compared with the un-modified nanoparticles (NPs). The in vivo retention of injected RNPs with hydrogel was obviously longer than that of NPs with hydrogel. The release of tacrolimus from T-RNPs + Gel could be sustained for 28 days. Compared with free tacrolimus-loaded hydrogel, the immune responses were significantly reduced and the survival of EPCs was greatly improved both in vitro and in vivo. The results of histological evaluation, including accumulation of immune cells and deposition of anti-graft antibodies, further revealed significantly lessened immune rejection in T-RNPs-anchoring hydrogel group compared with other groups. In pharmacodynamics study, the scaffold laden with EPCs was applied to treat hind limb ischemia in mice and significantly promoted the blood perfusion (~91 % versus ~36 % in control group). Conclusion: The nanoparticle-anchoring hydrogel scaffold is promising for localized immunosuppressant release, thereby can enhance the survival of transplanted cells and finally lead to successful tissue regeneration.

Cell-Permeable, MMP-2 Activatable, Nickel Ferrite and His-Tagged Fusion Protein Self-Assembled Fluorescent Nanoprobe for Tumor Magnetic-Targeting and Imaging.

Matrix metalloproteinases (MMPs) activatable imaging probe has been explored for tumor detection. However, activation of the probe is mainly done in the extracellular space without intracellular uptake of the probe for more sensitivity. Although cell-penetrating peptides (CPPs) have been demonstrated to enable intracellular delivery of the imaging probe, they nevertheless encounter off-target delivery of the cargos to normal tissues. Herein, we have developed a dual MMP-2-activatable and tumor cell-permeable magnetic nanoprobe to simultaneously achieve selective and intracellular tumor imaging. This novel imaging probe was constructed by self-assembling a hexahistidine-tagged (His-tagged) fluorescent fusion protein chimera and nickel ferrite nanoparticles via a chelation mechanism. The His-tagged fluorescent protein chimera consisted of a red fluorescent protein mCherry that acted as the fluorophore, the low-molecular-weight protamine peptide as the CPP, and the MMP-2 cleavage sequence fused with the hexahistidine tag, whereas the nickel ferrite nanoparticles functioned as the His-tagged protein binder and also the fluorescent quencher. Both in vitro and in vivo results revealed that this imaging probe would not only remain nonpermeable to normal tissues, thereby offsetting the nonselective cellular uptake, but was also suppressed of fluorescent signals during magnetic tumor-targeting in the circulation, primarily because of the masking of the CPP activity and quenching of the fluorophore by the associated NiFe2O4 nanoparticles. However, these properties were recovered or "turned on" by the action of tumor-associated MMP-2 stimuli, leading to cell penetration of the nanoprobes as well as fluorescence restoration and visualization within the tumor cells. In this regard, the presented tumor-activatable and cell-permeable system deems to be an appealing platform to achieve selective tumor imaging and intracellular protein delivery. Its impact is therefore significant, far-reaching, and wide-spread.

Heparin-Regulated Prodrug-Type Macromolecular Theranostic Systems for Cancer Therapy.

Heparin is a kind of naturally occurring polymer with excellent biocompatibility and solubility. It is characterized by dense of negative charge, higher than any endogenous components. Heparin can bind with various cationic peptides and proteins, thereby providing a useful noncovalent linkage for building a drug delivery system. As a case in point, heparin/cell-penetrating peptides (CPP) interaction is strong, and remains stable in vivo. They can be used to modify different proteins, respectively, and subsequently, by simply mixing the modified proteins, a protein-protein conjugate can be form via the stable heparin/CPP linkage. This linkage could not be broken unless addition of protamine that bears higher cationic charge density than CPP, and CPP thus can be substituted and released. Of note, heparin is a potent antagonist of CPP, and their binding naturally inhibits CPP-mediated drug cell penetration. Based on this method, we developed a heparin-regulated macromolecular prodrug-type system, termed ATTEMPTS, for drug targeting delivery. In this review article, we mainly summary the application of ATTEMPTS in delivery of various macromolecular drugs for cancer therapy, and also introduce the heparin-regulated nanoprobes for tumor imaging.

High-Yield Synthesis of Monomeric LMWP(CPP)-siRNA Covalent Conjugate for Effective Cytosolic Delivery of siRNA.

Because of the unparalleled efficiency and universal utility in treating a variety of disease types, siRNA agents have evolved as the future drug-of-choice. Yet, the inability of the polyanionic siRNA macromolecules to cross the cell membrane remains as the bottleneck of possible clinical applications. With the cell penetrating peptides (CPP) being discovered lately, the most effective tactic to achieve the highest intracellular siRNA delivery deems to be by covalently conjugating the drug to a CPP; for instance, the arginine-rich Tat or low molecular weight protamine (LMWP) peptides. However, construction of such a chemical conjugate has been referred by scientists in this field as the "Holy Grail" challenge due to self-assembly of the cationic CPP and anionic siRNA into insoluble aggregates that are deprived of the biological functions of both compounds. Based on the dynamic motion of PEG, we present herein a concise coupling strategy that is capable of permitting a high-yield synthesis of the cell-permeable, cytosol-dissociable LMWP-siRNA covalent conjugates. Cell culture assessment demonstrates that this chemical conjugate yields by far the most effective intracellular siRNA delivery and its corresponded gene-silencing activities. This work may offer a breakthrough advance towards realizing the clinical potential of all siRNA therapeutics and, presumably, most anionic macromolecular drugs such as anti-sense oligonucleotides, gene compounds, DNA vectors and proteins where conjugation with the CPP encounters with problems of aggregation and precipitation. To this end, the impact of this coupling technique is significant, far-reaching and wide-spread.

Drug depot-anchoring hydrogel: A self-assembling scaffold for localized drug release and enhanced stem cell differentiation.

Localized and long-term delivery of growth factors has been a long-standing challenge for stem cell-based tissue engineering. In the current study, a polymeric drug depot-anchoring hydrogel scaffold was developed for the sustained release of macromolecules to enhance the differentiation of stem cells. Self-assembling peptide (RADA16)-modified drug depots (RDDs) were prepared and anchored to a RADA16 hydrogel. The anchoring effect of RADA16 modification on the RDDs was tested both in vitro and in vivo. It was shown that the in vitro leakage of RDDs from the RADA16 hydrogel was significantly less than that of the unmodified drug depots (DDs). In addition, the in vivo retention of injected hydrogel-incorporated RDDs was significantly longer than that of hydrogel-incorporated unmodified DDs. A model drug, vascular endothelial growth factor (VEGF), was encapsulated in RDDs (V-RDDs) as drug depot that was then anchored to the hydrogel. The release of VEGF could be sustained for 4weeks. Endothelial progenitor cells (EPCs) were cultured on the V-RDDs-anchoring scaffold and enhanced cell proliferation and differentiation were observed, compared with a VEGF-loaded scaffold. Furthermore, this scaffold laden with EPCs promoted neovascularization in an animal model of hind limb ischemia. These results demonstrate that self-assembling hydrogel-anchored drug-loaded RDDs are promising for localized and sustained drug release, and can effectively enhance the proliferation and differentiation of resident stem cells, thus lead to successful tissue regeneration.

Tandem-multimeric F3-gelonin fusion toxins for enhanced anti-cancer activity for prostate cancer treatment.

Despite significant progress in prostate cancer treatment, yet, it remains the leading diagnosed cancer and is responsible for high incidence of cancer related deaths in the U.S. Because of the insufficient efficacy of small molecule anti-cancer drugs, significant interest has been drawn to more potent macromolecular agents such as gelonin, a plant-derived ribosome inactivating protein (RIP) that efficiently inhibits protein translation. However, in spite of the great potency to kill tumor cells, gelonin lacks ability to internalize tumor cells and furthermore, cannot distinguish between tumor and normal cells. To address this challenge, we genetically engineered gelonin fusion proteins with varied numbers of F3 peptide possessing homing ability to various cancer cells and angiogenic blood vessels. The E. coli produced F3-gelonin fusion proteins possessed equipotent activity to inhibit protein translation in cell-free protein translation systems to unmodified gelonin; however, they displayed higher cell uptake that led to significantly augmented cytotoxicity. Compared with gelonin fusion with one F3 peptide (F3-Gel), tandem-multimeric F3-gelonins showed even greater cell internalization and tumor cell killing ability. Moreover, when tested against LNCaP s.c. xenograft tumor bearing mice, more significant tumor growth inhibition was observed from the mice treated with tandem-multimeric F3-gelonins. Overall, this research demonstrated the potential of utilizing tandem multimeric F3-modified gelonin as highly effective anticancer agents to overcome the limitations of current chemotherapeutic drugs.

A novel strategy to achieve effective drug delivery: exploit cells as carrier combined with nanoparticles.

Cell-mediated drug delivery systems employ specific cells as drug vehicles to deliver drugs to targeted sites. Therapeutics or imaging agents are loaded into these cells and then released in diseased sites. These specific cells mainly include red blood cells, leukocytes, stem cells and so on. The cell acts as a Trojan horse to transfer the drug from circulating blood to the diseased tissue. In such a system, these cells keep their original properties, which allow them to mimic the migration behavior of specific cells to carry drug to the targeted site after in vivo administration. This strategy elegantly combines the advantages of both carriers, i.e. the adjustability of nanoparticles (NPs) and the natural functions of active cells, which therefore provides a new perspective to challenge current obstacles in drug delivery. This review will describe a fundamental understanding of these cell-based drug delivery systems, and discuss the great potential of combinational application of cell carrier and NPs.