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Stem cell-based therapeutic approach provides a possible treatment for critical limb ischemia (CLI) by inducing revascularization and regenerating ischemic tissue. However, the clinical benefit is modest due to low cell survival and limited efficacy after transplantation. Cardiac-derived stem cells (CSCs) might be a novel cell source for CLI treatment owing to their superb endothelial differentiation potential and angiogenic paracrine functions. In this study, the angiogenic ability of CSCs was maximized by genetic engineering with constitutively active form of hypoxia-inducible factor-1α (CA-HIF-1α), resistant to oxygen-dependent degradation. CSCs transfected with CA-HIF-1α (CA-HIF-CSCs) promoted supplementary expression of proangiogenic factors including VEGF, bFGF, Ang-1 and PDGF-B, along with enhanced angiogenic function including migratory effect, tube formation and endothelial differentiation potential. In the mouse CLI model, CA-HIF-CSCs transplanted into the ischemic region using fibrin gel as cell delivery vehicle, improved blood perfusion and limb functional recovery with minimal incidence of foot necrosis and limb loss by promoting new vessel formation. Histological evidence further confirmed that CA-HIF-CSC/gel treatment markedly alleviated muscle degeneration and fibrosis. CSCs genetically engineered with constitutively active HIF-1α provide a novel therapeutic modality in CLI combining stem cell and gene therapy.

Ribosome-inactivating proteins (RIPs) are potent protein toxins for cancer therapy, and they have strong ability to inhibit protein synthesis and induce cell death via inactivation of ribosomes in eukaryotic cells. However, the delivery of RIPs has been a challenging task due to their large molecular weight and lack of targeting property. Low molecular weight protamine (LMWP), a transmembrane peptide, has been proved to effectively promote transmembrane transportation, whereas the enzyme-activatable system can enhance the specificity by enhancing the tumor drug concentration through enzymatic reaction. We herein constructed a self-assembly and stimuli-responsive fusion gelonin delivery system. Gelonin, a typical RIP protein, was assembled with nickel ferrite nanoparticles by self-assembling between hexa-histidine tag (His-tagged) and nickel ions. Both in vitro and in vivo results indicated that the magnetic nanoparticle carriers and the applied linkers did not damage the pharmaceutical effect of gelonin, and the whole drug delivery system showed good biocompatibility, sensitive selectivity, and significantly enhanced cytotoxic activity. This in turn presented theranostic nanoparticles as efficient delivery vehicle for clinical use.

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.

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.

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.

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.

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., Graphical abstract A dual functional system was prepared based on dextrin-coated silver nanoparticles (AgNPs) which were further attached with iron oxide nanoparticles and cell penetrating peptide (Tat). The dual functionalized Tat-FeAgNPs could sufficiently target tumor by magnetism assisted tumor retention and Tat helped tissue penetration.fx1

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.

Inefficient cellular uptake and intracellular drug release at the tumor site are two major obstacles limiting the antitumor efficacy of nanoparticle delivery systems. To overcome both problems, we designed a smart nanoparticle that undergoes phase transition in a tumor microenvironment (TME). The smart nanoparticle is generated using a lipid-polypetide hybrid nanoparticle, which comprises a PEGylated lipid monolayer shell and a pH-sensitive hydrophobic poly-l-histidine core and is loaded with the antitumor drug doxorubicin (DOX). The smart nanoparticle undergoes a two-step phase transition at two different pH values in the TME: (i) At the TME (pHe: 7.0-6.5), the smart nanoparticle swells, and its surface potential turns from negative to neutral, facilitating the cellular uptake; (ii) After internalization, at the acid endolysosome (pHendo: 6.5-4.5), the smart nanoparticle dissociates and induces endolysosome escape to release DOX into the cytoplasm. In addition, a tumor-penetrating peptide iNRG was modified on the surface of the smart nanoparticle as a tumor target moiety. The in vitro studies demonstrated that the iNGR-modified smart nanoparticles promoted cellular uptake in the acidic environment (pH 6.8). The in vivo studies showed that the iNGR-modified smart nanoparticles exerted more potent antitumor efficacy against late-stage aggressive breast carcinoma than free DOX. These data suggest that the smart nanoparticles may serve as a promising delivery system for sequential uptake and intracellular drug release of antitumor agents. The easy preparation of these smart nanoparticles may also have advantages in the future manufacture for clinical trials and clinical use.

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 NiFe

Immune checkpoint therapy encounters significant challenges in clinic, including low response rate, acquired resistance and immune-related adverse events. Combination immunotherapy targeting multiple independent but complementary pathways in immune evasion has the potential to enhance therapeutic efficacy. Herein, a combination therapeutic strategy that dually inhibiting FGL1, a recently discovered main ligand for immune checkpoint LAG-3, and TGF-β1, an immunosuppressive cytokine, is firstly reported for colorectal cancer immunotherapy by blocking immune checkpoint and modulating tumor microenvironment simultaneously. We established a cRGD-modified exosome with high siFGL1 and siTGF-β1 loading efficiency (cRGD-Exo/siMix) to realize the co-silence of FGL1 and TGF-β1. The constructed cRGD-Exo/siMix showed a significant anti-tumor effect both in vitro and in vivo. Analysis of the tumor immune microenvironment demonstrated an increased number of tumor infiltration CD8+ T cells while a decreased number of immunosuppressive cells, implying that this therapeutic approach boosted anti-tumor immunity by reshaping the tumor microenvironment. This work provides a new strategy for siRNA delivery and its applications in combined cancer immunotherapy.

Magnetic iron oxide nanoparticles (MIONs) have received much attention due to their unique properties such as ferromagnetic and superparamagnetic characters. These magnetic properties enable the broad use of MIONs in biomedical applications including magnetic resonance imaging (MRI), magnetically guided delivery, and hyperthermal therapy. In particular, magnetic field guided delivery systems have shown promising potential in the development of targeted drug delivery systems for brain tumors. This system facilitates the extravasation and accumulation of MIONs within the brain tumor under external magnetic field. However, the practical use of MIONs is highly limited due to the large physical size of MIONs required for the sufficient retention and accumulation of particles in the brain tumor. This study aims to enhance the accumulation and retention of MIONs in the brain tumor by in situ formation of large clusters of MIONs. To achieve this goal, MIONs with core size of 100 nm were modified with free thiol end groups by conjugating bi-functional poly(ethylene glycol) (NHS-PEG-SH). It is expected that the prepared MIONs-PEG-SH remain stable during the systemic circulation. When the circulating MIONs-PEG-SH are exposed to the external magnetic field applied to the brain tumor, the local concentration of MIONs-PEG-SH can be increased and subsequent interactions among MIONs induce a disulfide bond formation. As a result, in situ formation of the large clusters of MIONs allows enhanced accumulation and retention of MIONs in a rat brain tumor model. Moreover, when doxorubicin is loaded onto the MIONs, the biodistribution of doxorubicin at brain tumor site is highly enhanced, suggesting their potential use in theranostic applications.

An anticancer drug delivery system consisting of DNA nanoparticles synthesized by rolling circle amplification (RCA) was developed for prostate cancer membrane antigen (PSMA) targeted cancer therapy. The template of RCA was a DNA oligodeoxynucleotide coded with PSMA-targeted aptamer, drug-loading domain, primer binding site and pH-sensitive spacer. Anticancer drug doxorubicin, as the model drug, was loaded into the drug-loading domain (multiple GC-pair sequences) of the DNA nanoparticles by intercalation. Due to the integrated pH-sensitive spacers in the nanoparticles, in an acidic environment, the cumulative release of doxorubicin was far more than the cumulative release of the drug in the normal physiological environment. In cell uptake experiments, treated with doxorubicin loaded DNA nanoparticles, PSMA-positive C4-2 cells could take up more doxorubicin than PSMA-null PC-3 cells. The prepared DNA nanoparticles showed the potential as drug delivery system for PSMA targeting prostate cancer therapy.

While drug resistance appears to be an inevitable problem of an increasing number of anticancer drugs in monotherapy, combination drug therapy has become a prosperous method to reduce the administered total drug dosages as well as overcome the drug resistance of carcinoma cells. Curcumin, considered to possess multifaceted roles in cancer treatment according to its multiple anti-neoplastic mechanisms as a depressor of chemoresistance, can significantly facilitate its anti-cancer functions and improve therapeutic effects via combination usage with a variety of other drugs with different reaction mechanisms. To explore this possibility, four anti-cancer chemotherapeutic agents that all possess a certain degree of drug resistance problems, including three tyrosine kinase inhibitors (erlotinib, sunitinib and sorafenib) that are acting on different cell pathways and a typical anticancer drug doxorubicin, were combined with curcumin individually to examine the synergistic anti-tumor effect both in vitro and in vivo. Results revealed that sunitinib combined with curcumin at the molar ratio of 0.46 yielded the most potent synergistic effect in vitro, and was therefore chosen for further animal evaluation. To further enhance the anticancer effect, bovine serum albumin (BSA) nanoparticles were utilized as a carrier to deliver the selected drug combination in situ. Preliminary in vivo findings confirmed our hypothesis of being able to maintain a similar injected drug ratio for prolonged time periods in tested animals by our approach, thereby maximizing the therapeutic potency yet minimizing the toxicity of these drugs. This work could open up a new avenue on combination drug therapy and realization the clinical utility of such drugs.

Although significant progress has been achieved, effective oral delivery of protein drugs such as insulin by nanoparticle-based carrier systems still faces certain formidable challenges. Considerable amount of protein drug is released from the nanoparticles (NPs) in the gastrointestinal (GI) tract. Because of their low permeability through the intestinal mucosa, the released protein would be soon degraded by the large amount of proteases in the GI tract. Herein, we report an oral insulin delivery system that can overcome the above-mentioned problems by mucoadhesive NPs (MNPs) loaded with cell penetrating peptide-linked insulin conjugates. On one hand, after conjugation with low molecular weight protamine (LMWP), a cell penetrating peptide (CPP), insulin showed greatly improved permeability through intestinal mucus layer and epithelia. On the other hand, the mucoadhesive N-trimethyl chitosan chloride-coated PLGA nanoparticles (MNPs) that were loaded with conjugates enhanced the retention in the intestinal mucus layer. By adopting this delivery strategy, the LMWP-insulin conjugates released from the MNPs could be deprived from enzymatic degradation, due to the short distance in reaching the epithelia and the high permeation of the conjugates through epithelia. The oral delivery system of insulin designed by us showed a long-lasting hypoglycemia effect with a faster onset in diabetic rats. The pharmacological availability of orally delivered conjugates-loaded MNPs was 17.98±5.61% relative to subcutaneously injected insulin solution, with a 2-fold higher improvement over that by MNPs loaded with native insulin. Our results suggested that conjugation with CPP followed by encapsulation in MNPs provides an effective strategy for oral delivery of macromolecular therapeutics.

Brain delivery of macromolecular therapeutics (e.g., proteins) remains an unsolved problem because of the formidable blood–brain barrier (BBB). Although a direct pathway of nose-to-brain transfer provides an answer to circumventing the BBB and has already been intensively investigated for brain delivery of small drugs, new challenges arise for intranasal delivery of proteins because of their larger size and hydrophilicity. In order to overcome the barriers and take advantage of available pathways (e.g., epithelial tight junctions, uptake by olfactory neurons, transport into brain tissues, and intra-brain diffusion), a low molecular weight protamine (LMWP) cell-penetrating peptide was utilized to facilitate nose-to-brain transport. Cell-penetrating peptides (CPP) have been widely used to mediate macromolecular delivery through many kinds of biobarriers. Our results show that conjugates of LMWP–proteins are able to effectively penetrate into the brain after intranasal administration. The CPP-based intranasal method highlights a promising solution for protein therapy of brain diseases., Graphical abstract The low molecular weight protamine (LMWP) cell-penetrating peptide is utilized to mediate proteins for nose-to-brain transport. The conjugates of LMWP–proteins can effectively penetrate into the brains by noninvasive intranasal administration. The cell penetrating peptide-based intranasal method highlights a promising solution for protein therapy for brain diseases. fx1

Toxic gene therapy (or suicidal gene therapy) is gaining enormous interest, specifically for the treatment of cancer. The success of this therapy lies in several crucial factors, including the potency of gene products to kill the transfected tumor cells and the transfection ability of the transfection vehicles. To address the potency problem, in the present study, we engineered two separate mammalian transfection plasmids (pSAP and pGEL) containing genes encoding ribosome inactivating proteins (RIPs), gelonin and saporin. After the successful preparation and amplification of the plasmids, they were tested on various cancer cell lines (HeLa, U87, 9L, and MDA-MB-435) and a noncancerous cell line (293 HEK) using polyethyleneimine (PEI) as the transfection agent. Transfection studies performed under varying gene concentration, incubation time, and gene-to-PEI ratios revealed that, compared to the treatment of pGFP (GFP expression plasmid)/PEI, both pGEL/PEI and pSAP/PEI complexes could induce significantly augmented cytotoxic effects at only 2 μg/mL gene concentration. Importantly, these cytotoxic effects were observed universally in all tested cancer cell lines. Overall, this study demonstrated the potential of pGEL and pSAP as effective gene candidates for the toxic gene-based cancer therapy.

Multi-core, rattle-type Ag@silica nanoparticles with different silver loadings were prepared via selective etching methods. A series of characterizations revealed that multiple Ag cores were uniformly distributed inside the silica shell and that the silver element was dominantly metallic silver and part silver bromide; with a ratio of about 6:1. Silver nanoparticles immobilized by silica nanocapsules more freely interacted with 4-nitrophenol due to the highly dispersed nanosized Ag cores, the special capsule structure, and high silver loading. Furthermore, the cationic interior of the silica capsules may serve as pools to preferentially concentrate anionic 4-nitrophenol while pumping out the 4-aminophenol. In line with these effects, Ag@silica displayed the highest efficiency of catalytic activity during 4-NP reduction. The kinetic rate constant was one or two orders of magnitude larger than those of other types of catalyst. Meanwhile, Ag@silica was stable, recyclable, and maintains its activity even after 20 reaction cycles, and is thus a candidate for 4-NP decontamination.

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.

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.

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.

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.

Theranostic platform, which is equipped with both diagnostic and therapeutic functions, is a promising approach in cancer treatment. From various nanotheranostics studied, iron oxide nanoparticles have advantages since IONPs have good biocompatibility and spatial imaging capability. This review is focused on the IONP-based nanotheranostics for cancer imaging and treatment. The most recent progress for applications of IONP nanotheranostics is summarized, which includes IONP-based diagnosis, magnetic resonance imaging (MRI), multimodal imaging, chemotherapy, hyperthermal therapy, photodynamic therapy, and gene delivery. Future perspectives and challenges are also outlined for the potential development of IONP based theranostics in clinical use.

Tumor-associated proteases (TAPs), such as legumain, are actively involved in cancer progression; they have been used as biomarkers for diagnosis, prognosis, and drug targeting. As a result, in-vivo detection and trafficking of TAPs have attracted a great deal of attention. TAP-specific probes for in-vivo imaging, however, remain rare. A TAP-responsive hybrid nanoprobe system based on quantum dots (QD) and the fluorescence resonance energy transfer (FRET) effect is presented for the detection of legumain (asparaginyl endopeptidase), which is overexpressed in many tumors. A novel hybrid construction method is developed for fabricating the nanoprobe, by which the strong heparin–protamine affinity is used for conjugation. The hybrid comprises two components: 1) low-molecular-weight heparin (LH)-modified QD, and 2) low-molecular-weight protamine (LMWP)-conjugated fluorescence quencher QSY21, through a legumain-cleavable linker. The hybrid nanoprobe (i.e., a FRET system) is self-assembled via the LH–LMWP affinity. The linker between LMWP and QSY21 is selectively cleaved by legumain, leading to QSY21 detachment and fluorescence recovery in the tumor. In-vivo imaging is successfully achieved in the colon tumor mouse model. Importantly, such a hybrid nanoprobe system is adaptable for the detection of other TAPs (e.g., matrix metalloproteinase -2) by using an established, corresponding substrate–peptide linker, thereby offering a universal platform for TAP detection and tumor imaging.

Protein toxins, such as gelonin, are highly desirable anti-cancer drug candidates due to their unparalleled potency and repetitive reaction mechanism in inhibiting protein translation. However, for its potential application in cancer therapy, there remains the cell membrane barrier that allows permeation of only small molecules, which must be overcome. To address this challenge, we conjugated gelonin with a protein transduction domain (PTD), the TAT peptide, via genetic recombination. The chimeric TAT-gelonin fusion protein (TAT-Gel) retained equipotent N-glycosidase activity yet displayed greater cell uptake than unmodified recombinant gelonin (rGel), thereby yielding a significantly augmented cytotoxic activity. Remarkably, TAT-Gel displayed up to 177-fold lower IC₅₀ (avg. 54.3 nM) than rGel (avg. IC₅₀ : 3640 nM) in tested cell lines. This enhanced cytotoxicity, however, also raised potential toxicity concerns due to the non-selectivity of PTD in its mediated cell transduction. To solve this problem, we investigated the plausibility of regulating the cell transduction of TAT-Gel via a reversible masking using heparin and protamine. Here, we demonstrated, both in vitro and in vivo, that the cell transduction of TAT-Gel can be completely curbed with heparin and yet this heparin block can be efficiently reversed by the addition of protamine. This reversible tight regulation of the cell transduction of TAT-Gel by heparin and protamine sheds light of possible application of TAT-Gel in achieving a highly effective yet safe drug therapy for the treatment of tumors.

Nutrient transporters have been explored for biomimetic delivery targeting the brain. The albumin-binding proteins (e.g., SPARC and gp60) are overexpressed in many tumors for transport of albumin as an amino acid and an energy source for fast-growing cancer cells. However, their application in brain delivery has rarely been investigated. In this work, SPARC and gp60 overexpression was found on glioma and tumor vessel endothelium; therefore, such pathways were explored for use in brain-targeting biomimetic delivery. We developed a green method for blood-brain barrier (BBB)-penetrating albumin nanoparticle synthesis, with the capacity to coencapsulate different drugs and no need for cross-linkers. The hydrophobic drugs (i.e., paclitaxel and fenretinide) yield synergistic effects to induce albumin self-assembly, forming dual drug-loaded nanoparticles. The albumin nanoparticles can penetrate the BBB and target glioma cells via the mechanisms of SPARC- and gp60-mediated biomimetic transport. Importantly, by modification with the cell-penetrating peptide LMWP, the albumin nanoparticles display enhanced BBB penetration, intratumoral infiltration, and cellular uptake. The LMWP-modified nanoparticles exhibited improved treatment outcomes in both subcutaneous and intracranial glioma models, with reduced toxic side effects. The therapeutic mechanisms were associated with induction of apoptosis, antiangiogenesis, and tumor immune microenvironment regulation. It provides a facile method for dual drug-loaded albumin nanoparticle preparation and a promising avenue for biomimetic delivery targeting the brain tumor based on combination 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.

For the past 20 years, we have witnessed an unprecedented and, indeed, rather miraculous event of how cell-penetrating peptides (CPPs), the naturally originated penetrating enhancers, help overcome the membrane barrier that has hindered the access of bio-macromolecular compounds such as genes and proteins into cells, thereby denying their clinical potential to become potent anti-cancer drugs. By taking the advantage of the unique cell-translocation property of these short peptides, various payloads of proteins, nucleic acids, or even nanoparticle-based carriers were delivered into all cell types with unparalleled efficiency. However, non-specific CPP-mediated cell penetration into normal tissues can lead to widespread organ distribution of the payloads, thereby reducing the therapeutic efficacy of the drug and at the same time increasing the drug-induced toxic effects. In view of these challenges, we present herein a review of the new designs of CPP-linked vehicles and strategies to achieve highly effective yet less toxic chemotherapy in combating tumor oncology.

Successful development of a new drug is prohibitively expensive, and is estimated to cost approximately $100—500 million US dollars for a single clinical drug. Yet, a newly developed drug can only enjoy its patent protection for 18 years, meaning that after this protected time period, any company can manufacture this product and thus the profit generated by this drug entity would reduce dramatically. Most critically, once a drug is being synthesized, its physical, chemical, and biological attributes such as bioavailability and in vivo pharmacokinetics are all completely fixed and cannot be changed. In principal and practice, only the application of an appropriately designed drug delivery system (DDS) is able to overcome such limitations, and yet the cost of developing a novel drug delivery system is less than 10% of that of developing a new drug. Because of these reasons, the new trend in pharmaceutical development has already begun to shift from the single direction of developing new drugs in the past to a combined mode of developing both new drugs and innovative drug delivery systems in this century. Hence, for developing countries with relatively limited financial resources, a smart strategic move would be to focus on the development of new DDS, which has a significantly higher benefit/risk ratio when comparing to the development of a new drug.

Although oral delivery of insulin offers a number of unmatched advantages, it nevertheless is beset by the poor permeability of insulin molecules through the epithelial cell membranes of the intestinal mucosal layer. We previously reported the development of low molecular weight protamine (LMWP) as a nontoxic yet potent cell penetrating peptide, of which via covalent linkage was capable of translocating protein cargos through the membranes of almost all cell types. It is therefore hypothesized that LMWP could be practically employed as a safe and effective tool to deliver insulin across the intestinal mucosal membrane, thereby augmenting its absorption through the GI tract. However, formulating 1:1 monomeric insulin/LMWP conjugate presents a tall order of challenge, as the acidic insulin and basic LMWP would automatically form tight aggregates through electrostatic interactions. In this paper, we developed an innovative conjugation strategy to solve this problem, by using succinimidyl-[(N-maleimidopropionamido)-polyethyleneglycol] ester (NHS-PEG-MAL) as an intermediate cross-linker during the coupling process. Both SDS-PAGE and MALDI-TOF mass spectroscopy confirmed the formation of a homogeneous, monomeric (1:1 ratio) insulin/LMWP conjugate without encountering the conventional problem of substrate aggregation. Cell culture studies demonstrated that transport of the Insulin-PEG-LMWP conjugate across the intestinal mucosal monolayer was augmented by almost five folds compared to native insulin. Furthermore, results from the in situ loop absorption tests in rats showed that systemic pharmacological bioavailability of insulin was significantly enhanced after its conjugation with LMWP. Overall, the presented chemical conjugation with LMWP could offer a reliable and safe means to improve the intestinal permeability of therapeutic peptides/proteins, shedding light of the possibility for their effective oral delivery.

A novel PEGylated and heparinized magnetic iron oxide nano-platform (DNPH) was synthesized for simultaneous magnetic resonance imaging (MRI) and tumor targeting.Starch-coated magnetic iron oxide nanoparticles ("D") were crosslinked, aminated (DN) and then simultaneously PEGylated and heparinized with different feed ratios of PEG and heparin (DNPH1-4). DNPH products were characterized by Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and superconducting quantum interference device (SQUID). The magentic targeting of DNPH3, with appropriate amounts of conjugated PEG and heparin, in a mouse 9L-glioma subcutaneous tumor model was confirmed by magnetic resonance imaging (MRI)/electron spin resonance (ESR).DNPH3 showed long circulating properties in vivo (half-life8 h, more than 60-fold longer than that of parent D) and low reticuloendothelial system (RES) recognition in liver and spleen. Protamine, a model cationic protein, was efficiently loaded onto DNPH3 with a maximum loading content of 26.4 μg/mg Fe. Magnetic capture of DNPH3 in tumor site with optimized conditions (I.D. of 12 mg/kg, targeting time of 45 min) was up to 29.42 μg Fe/g tissue (12.26% I.D./g tissue).DNPH3 showed the potential to be used as a platform for cationic proteins for simultaneous tumor targeting and imaging.

A well-defined, one-to-one conjugate between human serum albumin (HSA) and protamine was synthesized and characterized as a biocompatible carrier for macromolecules. In circulation, the conjugate will camouflage drug molecules upon complex formation, while liberating free drug at the desired location using a triggering mechanism. The N-terminus of protamine was thiolated and conjugated with the unpaired Cysteine-34 of HSA, and was purified by ion-exchange chromatography. The molecular weight of the conjugate was 70.8 kDa, confirming one-to-one conjugation between HSA (66.6 KDa) and protamine (4200 Da). Superimposed fluorescence spectra of native HSA and HSA–protamine conjugate indicated no conformational change around the Trp-214. The conjugate had marked reduction in hemolytic and cytotoxic properties compared to protamine. When therapeutic potential was tested using tissue plasminogen activator as a model drug, HSA–protamine conjugate suppressed the enzymatic activity by 65%, which was fully recovered by a triggering agent, heparin. The construct showed binding characteristics with activated platelets upon conjugation with a targeting peptide, demonstrating flexibility to introduce suitable homing moiety on the surface. The camouflaged construct retained triggered release property in human plasma condition. Overall, the conjugate has a good potential to serve as a biocompatible platform for macromolecular drugs. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 102A: 2481–2490, 2014.

One of the major hurdles to cure cancer lies in the low potency of currently available drugs, which could eventually be solved by using more potent therapeutic macromolecules, such as proteins or genes. However, although these macromolecules possess greater potency inside the cancer cells, the barely permeable cell membrane remains a formidable barrier to exert their efficacy. A widely used strategy is to use cell penetrating peptides (CPPs) to improve their intracellular uptake. Since the discovery of the first CPP, numerous CPPs have been derived from natural or synthesized products. Both in vitro and in vivo studies have demonstrated that those CPPs are highly efficient in transducing cargoes into almost all cell types. Therefore, to date, CPPs have been widely used for intracellular delivery of various cargoes, including peptides, proteins, genes, and even nanoparticles. In addition, recently, based on the successes of CPPs in cellular studies, their applications in vivo have been actively pursued. This review will focus on the advanced applications of CPP-based in vivo delivery of therapeutics (e.g., small molecule drugs, proteins, and genes). In addition, we will highlight certain updated applications of CPPs for intracellular delivery of nanoparticulate drug carriers, as well as several "smart" strategies for tumor targeted delivery of CPP-cargoes.

Oral insulin delivery has received the most attention in insulin formulations due to its high patient compliance and, more importantly, to its potential to mimic the physiologic insulin secretion seen in non-diabetic individuals. However, oral insulin delivery has two major limitations: the enzymatic barrier that leads to rapid insulin degradation, and the mucosal barrier that limits insulin’s bioavailability. Several approaches have been actively pursued to circumvent the enzyme barrier, with some of them receiving promising results. Yet, thus far there has been no major success in overcoming the mucosal barrier, which is the main cause in undercutting insulin’s oral bioavailability. In this review of our group’s research, an innovative silica-based, mucoadhesive oral insulin formulation with encapsulated-insulin/cell penetrating peptide (CPP) to overcome both enzyme and mucosal barriers is discussed, and the preliminary and convincing results to confirm the plausibility of this oral insulin delivery system are reviewed. In vitro studies demonstrated that the CPPinsulin conjugates could facilitate cellular uptake of insulin while keeping insulin’s biologic functions intact. It was also confirmed that low molecular weight protamine (LMWP) behaves like a CPP peptide, with a cell translocation potency equivalent to that of the widely studied TAT. The mucoadhesive properties of the produced silica-chitosan composites could be controlled by varying both the pH and composition; the composite consisting of chitosan (25 wt-%) and silica (75 wt-%) exhibited the greatest mucoadhesion at gastric pH. Furthermore, drug release from the composite network could also be regulated by altering the chitosan content. Overall, the universal applicability of those technologies could lead to development of a generic platform for oral delivery of many other bioactive compounds, especially for peptide or protein drugs which inevitably encounter the poor bioavailability issues.

With the hope of overcoming the serious side effects, great endeavor has been made in tumor-targeted chemotherapy, and various drug delivery modalities and drug carriers have been made to decrease systemic toxicity caused by chemotherapeutic agents. Scientists from home and abroad focus on the research of targeted microbubbles contrast agent, and the use of the targeted ultrasound microbubble contrast agent can carry gene drugs and so on to the target tissue, as well as mediated tumor cell apoptosis and tumor microvascular thrombosis block, etc., thus plays the role of targeted therapy. Recent studies have elucidated the mechanisms of drug release and absorption, however, much work remains to be done in order to develop a successful and optimal system. In this review, we summarized the continuing efforts in understanding the usage of the ultrasound triggered target microbubbles in cancer therapy, from release mechanism to preparation methods. The latest applications of ultrasound-triggered targeted microbubbles in cancer therapy, especially in gene therapy and antiangiogenic cancer therapy were discussed. Moreover, we concluded that as a new technology, ultrasound-triggered targeted microbubbles used as drug carriers and imaging agents are still energetic and are very likely to be translated into clinic in the near future.

Simvastatin (SIM), a drug commonly administered for the treatment of hypercholesterolemia, has been recently reported to induce bone regeneration/formation. In this study, we investigated the properties of hydrogel composed of gelatin-poly(ethylene glycol)-tyramine (GPT) as an efficient SIM delivery vehicle that can trigger osteogenic differentiation. Sustained delivery of SIM was achieved through its encapsulation in an injectable, biodegradable GPT-hydrogel. Cross-linking of the gelatin-based GPT-hydrogel was induced by the reaction of horse radish peroxidase and H(2)O(2). GPT-hydrogels of three different matrix stiffness, 1,800 (GPT-hydrogel1), 5,800 (GPT-hydrogel2), and 8,400 Pa (GPT-hydrogel3) were used. The gelation/degradation time and SIM release profiles of hydrogels loaded with two different concentrations of SIM, 1 and 3 mg/ml, were also evaluated. Maximum swelling times of GPT-hydrogel1, GPT-hydrogel2, and GPT-hydrogel3 were observed to be 6, 12, and 20 days, respectively. All GPT-hydrogels showed complete degradation within 55 days. The in vitro SIM release profiles, investigated in PBS buffer (pH 7.4) at 37°C, exhibited typical biphasic release patterns with the initial burst being more rapid with GPT-hydrogel1 compared with GPT-hydrogel3. Substantial increase in matrix metalloproteinase-13, osteocalcin expression levels, and mineralization were seen in osteogenic differentiation system using MC3T3-E1 cells cultured with GPT-hydrogels loaded with SIM in a dose-dependent manner. This study demonstrated that controlled release of SIM from a biodegradable, injectable GPT-hydrogel had a promising role for long-term treatment of chronic degenerative diseases such as disc degenerative disease.

The regulation of angiogenesis is an interesting area to consider for novel therapeutic approaches to rheumatoid arthritis (RA). Chemically modified heparins have been developed as possible candidates for angiogenesis inhibitor; however, they have a major clinical drawback in exhibiting poor oral bioavailability. Here, orally absorbable O-desulfated low molecular weight heparin (ODS-LMWH) derivatives were newly synthesized by conjugating 2-O- or 6-O-desulfated LMWH with deoxycholic acid (DOCA) or bisDOCA (a dimer of DOCA), and their physicochemical properties, antiangiogenic potency and pharmacokinetic profiles were assessed. After selecting the best candidate among those derivatives, its therapeutic efficacy on arthritis was investigated in a murine collagen antibody-induced arthritis (CAIA) model. ODS-LMWH derivatives significantly inhibited the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs) and basic fibroblast growth factor (bFGF)-induced angiogenesis in the Matrigel plug assay. Among all the compounds, 6ODS-LHbD showed the highest oral bioavailability in rats (19.3%). In the CAIA mouse model, 6ODS-LHbD (10 mg/kg, p.o., S.I.D.) significantly inhibited neovascularization in the joint, the increase of hind-paw thickness, and the structural damage in the bone. Therefore, 6ODS-LHbD would be a promising candidate for an orally active drug for the treatment of RA.

To investigate the applicability of fusion biotoxins combining pore-forming toxins (PFTs) and ribosome-inactivating proteins (RIPs) for the anti-cancer treatment.Membrane active PFTs tend to destabilize cell membranes of tumor cells, but lack a warhead inducing significant cause of cell death. Cell-impermeable RIPs possess a powerful warhead, yet not able to enter the tumor cells. To address these challenges for anti-tumor effects, we introduced a fusion strategy of conjugating melittin (a PFT) and gelonin (a type 1 RIP) via chemical and recombinant methods, followed by in vitro assays and in vivo animal studies.In vitro characterization results confirmed that the chimeric gelonin-melittin fusion proteins retained equivalent intrinsic activity to that of unmodified gelonin in inhibiting protein translation. However, chemically conjugated gelonin-melittin (cGel-Mel) and recombinant chimeric gelonin-melittin fusion (rGel-Mel) exhibited greater cell uptake, yielding a significantly enhanced cytotoxic activity over treatment of gelonin, melittin or physical mixture of gelonin and melittin. Remarkably, cGel-Mel and rGel-Mel displayed 32- and 10-fold lower IC50 than gelonin in the cell lines. The superior anti-tumor efficacy of multivalent cGel-Mel to monovalent rGel-Mel suggested that valency could be a crucial factor for the extent of melittin-mediated cell uptake. Tumoricidal effects observed from animal studies were in good accordance with our findings from the cellular assays.This study successfully demonstrated that fusion of biotoxins could provide a simple yet effective way to synergistically augment their anti-tumor activity.

Despite advances in surgery and drug discovery, brain tumors remain fatal diseases. Early detection and diagnosis of brain tumors is of great importance for improving treatment outcomes. Magnetic resonance imaging (MRI) is a prominent, clinically-relevant imaging modality because of its excellent tissue contrast resolution, direct multiplanar imaging and increased sensitivity to edema. MRI utility is further enhanced with the use of magnetic iron oxide nanoparticles, which can function as both a contrast agent for imaging and as a drug delivery vehicle for treating brain cancer. In this review, the principles of various imaging modalities for brain tumors are discussed with focus on monocrystalline iron oxide nanoparticle (MION)-based MRI contrast agents. A summary is given on the mechanism of contrast effect, magnetophoretic mobility and magnetic retention, and strategies to enhance tumor selectivity, increase spatial resolution and reduce nonspecific uptake of MION.

Folate-conjugated and thermo-responsive poly((N-isopropylacrylamide)-co- acrylamide-co-(octadecyl acrylate)-co-(folate-(polyethylene glycol)-(acrylic acid))) (P(NIPA-co-AAm-co-ODA-co-FPA)) micelles with mean diameter of about 60 nm and lower critical solution temperature (LCST) of about 39°C were synthesized by free radical random copolymerization. Single-factor tests of acrylamide and octadecyl acrylate were carried out to modulate micelles' LCST and diameter, respectively. LCST, diameter, and morphology of micelles were determined by UV-vis spectrophotometer, laser particle size analyzer, and transmittance electron microscope (TEM), respectively. Fluorescein was then used as a model drug to investigate the drug loading content of micelles. Micelles with maximum amount of octadecyl acrylate (180 mg) were found to yield drug loading content of 10.48%. Near infrared dye No.10 was chosen as the tracer to monitor micelles in vivo. The targeting behaviors of micelles in folate receptor positive Bel-7402 tumor bearing nude mice were assessed by a self-constructed near infrared imaging system. Results showed satisfactory targeting capability of the thermo-responsive micelles toward Bel-7402 tumors, and targeting accumulation could last for more than 96 h, enabling P(NIPA-co-AAm-co-ODA-co-FPA) micelles to function as a diagnostic reagent as well as a targeted tumor therapy.

The chemical conjugate of low molecular weight heparin with taurocholate (LHT7) was previously designed to offer anticancer activity while minimizing the anticoagulant activity. In the present study, we found that the systemic administration of LHT7 in nanolipoplex could substantially enhance tumor vasculature targeting and anticancer effects. Moreover, we found that co-delivery of LHT7 with suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, in nanolipoplex could provide synergistic antitumor effect. LHT7/SAHA nanolipoplex was formulated by encapsulating SAHA inside cationic liposomes, followed by complexation of negatively charged LHT7 onto the cationic surfaces of SAHA-loaded liposomes (SAHA-L). LHT7/SAHA nanolipoplex was positively charged with a mean diameter of 117.6 nm, and stable in serum. The nanolipoplex form of LHT7 could alter its pharmacokinetics and biodistribution. Compared to the free form of LHT7, LHT7 in the nanolipoplex showed 1.9-fold higher mean residence time, and higher tumor vasculature accumulation after its intravenous administration. LHT7/SAHA nanolipoplex showed highest antitumor efficacy in SCC-bearing mice, compared to LHT7, SAHA-L and sequential co-administration of LHT7 and SAHA-L. Consistent with the enhanced antitumor effect, the reduction of abnormal vessels in the tumor site was also the highest in the LHT7/SAHA nanolipoplex-treated group. These results suggested the potential of LHT7/SAHA nanolipoplex for enhanced tumor vasculature targeting, and the importance of nanolipoplex-mediated co-delivery with a histone deacetylase inhibitor for maximal anticancer effect.