Multifunctional elastin-like polypeptide nanocarriers for efficient miRNA delivery in cancer therapy.

BACKGROUND: The exogenous delivery of miRNA to mimic and restore miRNA-34a activity in various cancer models holds significant promise in cancer treatment. Nevertheless, its effectiveness is often impeded by challenges, including a short half-life, propensity for off-target accumulation, susceptibility to inactivation by blood-based enzymes, concerns regarding patient safety, and the substantial cost associated with scaling up. As a means of overcoming these barriers, we propose the development of miRNA-loaded Tat-A86 nanoparticles by virtue of Tat-A86's ability to shield the loaded agent from external environmental factors, reducing degradation and inactivation, while enhancing circulation time and targeted accumulation. RESULTS: Genetically engineered Tat-A86, featuring 16 copies of the interleukin-4 receptor (IL-4R)-binding peptide (AP1), Tat for tumor penetration, and an elastin-like polypeptide (ELP) for presenting target ligands and ensuring stability, served as the basis for this delivery system. Comparative groups, including Tat-E60 and A86, were employed to discern differences in binding and penetration. The designed ELP-based nanoparticle Tat-A86 effectively condensed miRNA, forming stable nanocomplexes under physiological conditions. The miRNA/Tat-A86 formulation bound specifically to tumor cells and facilitated stable miRNA delivery into them, effectively inhibiting tumor growth. The efficacy of miRNA/Tat-A86 was further evaluated using three-dimensional spheroids of lewis lung carcinoma (LLC) as in vitro model and LLC tumor-bearing mice as an in vivo model. It was found that miRNA/Tat-A86 facilitates effective cell killing by markedly improving miRNA penetration, leading to a substantial reduction in the size of LLC spheroids. Compared to other controls, Tat-A86 demonstrated superior efficacy in suppressing the growth of 3D cellular aggregates. Moreover, at equivalent doses, miRNA-34a delivered by Tat-A86 inhibited the growth of LLC cells in allograft mice. CONCLUSIONS: Overall, these studies demonstrate that Tat-A86 nanoparticles can deliver miRNA systemically, overcoming the basic hurdles impeding miRNA delivery by facilitating both miRNA uptake and stability, ultimately leading to improved therapeutic effects.

Development of elastin-like polypeptide for targeted specific gene delivery in vivo.

BACKGROUND: The successful deliveries of siRNA depend on their stabilities under physiological conditions because greater in vivo stability enhances cellular uptake and enables endosomal escape. Viral-based systems appears as most efficient approaches for gene delivery but often compromised in terms of biocompatibility, patient safety and high cost scale up process. Here we describe a novel platform of gene delivery by elastin-like polypeptide (ELP) based targeting biopolymers. RESULTS: For better tumor targeting and membrane penetrating characteristics, we designed various chimeric ELP-based carriers containing a cell penetrating peptide (Tat), single or multiple copies of AP1 an IL-4 receptor targeting peptide along with coding sequence of ELP and referred as Tat-A1E28 or Tat-A4V48. These targeted polypeptides were further analyzed for its ability to deliver siRNA (Luciferase gene) in tumor cells in comparison with non-targeted controls (Tat-E28 or E28). The positively charged amino acids of these polypeptides enabled them to readily complex with negatively charged nucleic acids. The complexation of nucleic acid with respective polypeptides facilitated its transfection efficiency as well as stability. The targeted polypeptides (Tat-A1E28 or Tat-A4V48) selectively delivered siRNA into tumor cells in a receptor-specific fashion, achieved endosomal and lysosomal escape, and released gene into cytosol. The target specific delivery of siRNA by Tat-A1E28 or Tat-A4V48 was further validated in murine breast carcinoma 4T1 allograft mice model. CONCLUSION: The designed delivery systems efficiently delivered siRNA to the target site of action thereby inducing significant gene silencing activity. The study shows Tat and AP1 functionalized ELPs constitute a novel gene delivery system with potential therapeutic applications.

Double-Chambered Ferritin Platform: Dual-Function Payloads of Cytotoxic Peptides and Fluorescent Protein.

Ferritin cage nanoparticles are promising platforms for targeted delivery of imaging and therapeutic agents. One of the main advantages of cage nanoparticles is the ability to display multiple functionalities through genetic modification so as to achieve desired therapeutic or diagnostic functions. Ferritin complexes formed from short ferritin (sFt) lacking the fifth helix can accommodate dual peptide and protein functionalities on N- and C-terminal sites in sFt monomers. The resulting double-chambered NanoCage (DCNC) offers the potential of dual activities; these activities are augmented by the avidity of the ligands, which do not impede each other's function. Here we demonstrated proof-of-concept of DCNCs, loading the tumor-targeting proapoptotic peptide CGKRK(KLAKLAK)2 onto the N-terminal chamber and green fluorescent protein (GFP) onto the C-terminal chamber. The resulting KLAK-sFt-GFP DCNCs were internalized into the human breast adenocarcinoma cell line MDA-MB-231 and induced apoptosis. These findings suggest that DCNCs containing various combinations of peptides and proteins could be applied as therapeutics in different diseases.

Immune Checkpoint-Blocking Nanocages Cross the Blood-Brain Barrier and Impede Brain Tumor Growth.

Glioblastoma (GBM) is the deadliest tumor of the central nervous system, with a median survival of less than 15 months. Despite many trials, immune checkpoint-blocking (ICB) therapies using monoclonal antibodies against the PD-1/PD-L1 axis have demonstrated only limited benefits for GBM patients. Currently, the main hurdles in brain tumor therapy include limited drug delivery across the blood-brain barrier (BBB) and the profoundly immune-suppressive microenvironment of GBM. Thus, there is an urgent need for new therapeutics that can cross the BBB and target brain tumors to modulate the immune microenvironment. To this end, we developed an ICB strategy based on the BBB-permeable, 24-subunit human ferritin heavy chain, modifying the ferritin surface with 24 copies of PD-L1-blocking peptides to create ferritin-based ICB nanocages. The PD-L1pep ferritin nanocages first demonstrated their tumor-targeting and antitumor activities in an allograft colon cancer model. Next, we found that these PD-L1pep ferritin nanocages efficiently penetrated the BBB and targeted brain tumors through specific interactions with PD-L1, significantly inhibiting tumor growth in an orthotopic intracranial tumor model. The addition of PD-L1pep ferritin nanocages to triple in vitro cocultures of T cells, GBM cells, and glial cells significantly inhibited PD-1/PD-L1 interactions and restored T-cell activity. Collectively, these findings indicate that ferritin nanocages displaying PD-L1-blocking peptides can overcome the primary hurdle of brain tumor therapy and are, therefore, promising candidates for treating GBM.

Dual Impact of IGF2 on Alveolar Stem Cell Function during Tobacco-Induced Injury Repair and Development of Pulmonary Emphysema and Cancer.

Pulmonary emphysema is a destructive inflammatory disease primarily caused by cigarette smoking (CS). Recovery from CS-induced injury requires proper stem cell (SC) activities with a tightly controlled balance of proliferation and differentiation. Here we show that acute alveolar injury induced by two representative tobacco carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene (N/B), increased IGF2 expression in alveolar type 2 (AT2) cells to promote their SC function and facilitate alveolar regeneration. Autocrine IGF2 signaling upregulated Wnt genes, particularly Wnt3, to stimulate AT2 proliferation and alveolar barrier regeneration after N/B-induced acute injury. In contrast, repetitive N/B exposure provoked sustained IGF2-Wnt signaling through DNMT3A-mediated epigenetic control of IGF2 expression, causing a proliferation/differentiation imbalance in AT2s and development of emphysema and cancer. Hypermethylation of the IGF2 promoter and overexpression of DNMT3A, IGF2, and the Wnt target gene AXIN2 were seen in the lungs of patients with CS-associated emphysema and cancer. Pharmacologic or genetic approaches targeting IGF2-Wnt signaling or DNMT prevented the development of N/B-induced pulmonary diseases. These findings support dual roles of AT2 cells, which can either stimulate alveolar repair or promote emphysema and cancer depending on IGF2 expression levels. SIGNIFICANCE: IGF2-Wnt signaling plays a key role in AT2-mediated alveolar repair after cigarette smoking-induced injury but also drives pathogenesis of pulmonary emphysema and cancer when hyperactivated.

Application of bioengineered elastin-like polypeptide-based system for targeted gene delivery in tumor cells.

Successful gene delivery depends on the entry of negatively charged DNAs and oligonucleotides across the various barriers of the tumor cells and localization into the nucleus for its transcription and protein translation. Here, we have reported a thermal responsive self-assemble and highly biocompatible, targeted ELP-based gene delivery system. These systems consist of cell-penetrating peptides, Tat and single or multiple repeats of IL-4 receptor targeting peptide AP-1 along the backbone of ELP. Cell-penetrating peptides were introduced for nuclear localization of genes of interest, AP-1 for targeting IL-4R highly expressed tumor cells and ELP for stable condensation favoring protection of nucleic acids. The designed multidomain fusion ELPs referred to as Tat-ELP, Tat-A1E28 and Tat-A4V48 were employed to generate formulation with pEGFP-N1. Profound formulation of stable complexes occurred at different molar ratios owing to electrostatic interactions of positively charged amino acids in polymers with negatively charged nucleic acids. Among the complexes, Tat-A4V48 containing four copies of AP-1 showed maximum complexation with pEGFP-N1 in lower molar ratio. The polymer-pEGFP complexes were further analyzed for its transfection efficiency in different cancer cell lines. Both the targeted polymers, Tat-A4V48 and Tat-A1E28 upon transfection displayed significant EGFP-expression with low toxicity in different cancer cells. Therefore, both Tat-A4V48 and Tat-A1E28 can be considered as novel transfection system for successful gene delivery with therapeutic applications.

Application of Bio-Active Elastin-like Polypeptide on Regulation of Human Mesenchymal Stem Cell Behavior.

Regenerative medicine using stem cells offers promising strategies for treating a variety of degenerative diseases. Regulation of stem cell behavior and rejuvenate senescence are required for stem cells to be clinically effective. The extracellular matrix (ECM) components have a significant impact on the stem cell's function and fate mimicking the local environment to maintain cells or generate a distinct phenotype. Here, human elastin-like polypeptide-based ECM-mimic biopolymer was designed by incorporating various cell-adhesion ligands, such as RGD and YIGSR. The significant effects of bioactive fusion ELPs named R-ELP, Y-ELP, and RY-ELP were analyzed for human bone-marrow-derived stem cell adhesion, proliferation, maintenance of stemness properties, and differentiation. Multivalent presentation of variable cell-adhesive ligands on RY-ELP polymers indeed promote efficient cell attachment and proliferation of human fibroblast cells dose-dependently. Similarly, surface modified with RY-ELP promoted strong mesenchymal stem cell (MSCs) attachment with greater focal adhesion (FA) complex formation at 6 h post-incubation. The rate of cell proliferation, migration, population doubling time, and collagen I deposition were significantly enhanced in the presence of RY-ELP compared with other fusion ELPs. Together, the expression of multipotent markers and differentiation capacity of MSCs remained unaffected, clearly demonstrating that stemness properties of MSCs were well preserved when cultured on a RY-ELP-modified surface. Hence, bioactive RY-ELP offers an anchorage support system and effectively induces stimulatory response to support stem cell proliferation.

Application of elastin-like polypeptide (ELP) containing extra-cellular matrix (ECM) binding ligands in regenerative medicine.

Extracellular matrix (ECM) molecules play an important role in regulating molecular signaling associated with proliferation, migration, differentiation, and tissue repair. The identification of new kinds of ECM mimic biomaterials to recapitulate critical functions of biological systems are important for various applications in tissue engineering and regenerative medicine. The use of human elastin derived materials with controlled biological properties and other functionalities to improve their cell-response was proposed. Herein, we reported genetic encoded synthesis of ELP (elastin-like polypeptide) containing ECM domains like RGD (integrin binding ligand) and YIGSR (laminin-selective receptor binding ligand) to regulate cell behaviour in more complex ways, and also better model natural matrices. Thermal responsiveness of the ELPs and structural conformation were determined to confirm its phase transition behaviour. The fusion ELPs derivatives were analysed for mechanical involvement of growth mechanism, regenerative, and healing processes. The designed fusion ELPs promoted fast and strong attachment of fibroblast cells. The fusion ELP derivatives enhanced the migration of keratinocyte cells which of crucial for wound healing. Together it provides a profound matrix for endothelial cells and significantly enhanced tube formation of HUVEC cells. Thus, strategy of using cell adhesive ELP biopolymer emphasizing the role of bioactive ELPs as next generation skin substitutes for regenerative medicine.

S100A14: A novel negative regulator of cancer stemness and immune evasion by inhibiting STAT3-mediated programmed death-ligand 1 expression in colorectal cancer.

BACKGROUND: Programmed death-ligand 1 (PD-L1) has functional roles in cancer stem-like cell (CSC) phenotypes and chemoresistance besides immune evasion. Chemotherapy is a common treatment choice for colorectal cancer (CRC) patients; however, chemoresistance limits its effectiveness of treatment. METHODS: We examined the role of S100A14 (SA14) in CRC by adopting PD-L1high subpopulations within CRC cell lines and patient tumours, by establishing PD-L1high chemoresistant CRC sublines through prolonged exposure to 5-fluorouracil/oxaliplatin-based chemotherapy in vitro and in vivo, and by analysing a public database. RESULTS: We identified a novel function of SA14 as a regulator of immune surveillance, major CSC phenotypes, and survival capacity under hostile microenvironments, including those harbouring chemotherapeutics, and as a prognostic biomarker in CRC. Mechanistically, SA14 inhibits PD-L1 expression by directly interacting with signal transducer and activator of transcription 3 (STAT3) and inducing its proteasome-mediated degradation. While gain-of-SA14 causes loss of PD-L1 expression and tumourigenic potential and sensitisation to chemotherapy-induced apoptosis in chemoresistant CRC cells, loss-of-SA14 causes increases in PD-L1 expression, tumourigenic potential, and chemoresistance in vitro and in vivo. We further show that a combinatorial treatment with chemotherapy and recombinant SA14 protein effectively induces apoptosis in PD-L1high chemoresistant CRC cells. CONCLUSIONS: Our results suggest that SA14-based therapy is an effective strategy to prevent tumour progression and that SA14 is a predictive biomarker for anti-PD-L1 immunotherapy and chemotherapy in combination.

Transforming growth factor-beta-induced protein (TGFBIp/beta ig-h3) activates platelets and promotes thrombogenesis.

Transforming growth factor-beta-induced protein (TGFBIp)/beta ig-h3 is a 68-kDa extracellular matrix protein that is functionally associated with the adhesion, migration, proliferation, and differentiation of various cells. The presence of TGFBIp in platelets led us to study the role of this protein in the regulation of platelet functions. Upon activation, platelet TGFBIp was released and associated with the platelets. TGFBIp mediates not only the adhesion and spread of platelets but also activates them, resulting in phosphatidylserine exposure, alpha-granule secretion, and increased integrin affinity. The fasciclin 1 domains of TGFBIp are mainly responsible for the activation of platelets. TGFBIp promotes thrombus formation on type I fibrillar collagen under flow conditions in vitro and induces pulmonary embolism in mice. Moreover, transgenic mice, which have approximately a 1.7-fold greater blood TGFBIp concentration, are significantly more susceptible to collagen- and epinephrine-induced pulmonary embolism than wild-type mice. These results suggest that TGFBIp, a human platelet protein, plays important roles in platelet activation and thrombus formation. Our findings will increase our understanding of the novel mechanism of platelet activation, contributing to a better understanding of thrombotic pathways and the development of new antithrombotic therapies.

Fibrinolytic nanocages dissolve clots in the tumor microenvironment, improving the distribution and therapeutic efficacy of anticancer drugs.

Fibrin, one of the components of the extracellular matrix (ECM), acts as a transport barrier within the core of tumors by constricting the blood vessels and forming clots, leading to poor intratumoral distribution of anticancer drugs. Our group previously developed a microplasmin-based thrombolytic ferritin nanocage that efficiently targets and dissolves clots without causing systemic fibrinolysis or disrupting hemostatic clots. We hypothesized that the thrombolytic nanocage-mediated degradation of fibrin clots in the tumor ECM can lead to enhanced intratumoral drug delivery, especially for nanosized anticancer drugs. Fibrin clot deposition worsens after surgery and chemotherapy, further hindering drug delivery. Moreover, the risk of venous thromboembolism (VTE) also increases. Here, we used thrombolytic nanocages with multivalent clot-targeting peptides and fibrin degradation enzymes, such as microplasmin, to dissolve fibrin in the tumor microenvironment and named them fibrinolytic nanocages (FNCs). These FNCs target tumor clots specifically and effectively. FNCs efficiently dissolve fibrin clots inside of the tumor vessels, suggesting that they can mitigate the risk of VTE in cancer patients. Coadministration of FNC and doxorubicin led to improved chemotherapeutic activity in a syngeneic mouse melanoma model. Furthermore, the FNCs increased the distribution of Doxil/doxorubicin nanoparticles within mouse tumors. These results suggest that fibrinolytic cotherapy might help improve the therapeutic efficacy of anticancer nanomedicines. Thus, microplasmin-based fibrinolytic nanocages are promising candidates for this strategy due to their hemostatic safety and ability to home in on the tumor.

Therapeutic Effect of IL-4 Receptor-Targeting Pro-Apoptotic Peptide (AP1-ELP-KLAK) in Glioblastoma Tumor Model.

BACKGROUND: Thermal-responsive self-assembled elastin-like polypeptide (ELP)-based nanoparticles are an emerging platform for controlled delivery of therapeutic peptides, proteins and small molecular drugs. The antitumor effect of bioengineered chimeric polypeptide AP1-ELP-KLAK containing an interleukin-4 receptor (IL-4R) targeting peptide and pro-apoptotic peptide (KLAKLAK) was evaluated in glioblastoma (GBM) in vitro and in vivo. METHODS AND RESULTS: Herein, the therapeutic effect of AP1-ELP-KLAK was tested in advanced, and less curable glioblastoma cells with higher expression of IL-4R. Glioblastoma cell lines stably expressing different reporter systems i.e., caspase-3 sensor (surrogate marker for cellular apoptosis) or effluc/enhanced firefly luciferase (cellular viability) were established to measure cell death non-invasively. Bioluminescence imaging (BLI) of D54/effluc and U97MG/effluc treated with AP1-ELP-KLAK exhibited higher cell death up to 2~3-fold than the control. Treatment with AP1-ELP-KLAK resulted in time-dependent increase of caspase-3 sensor BLI activity in D54/C cells and D54/C tumor-bearing mice. Intravenous injection of AP1-ELP-KLAK dramatically reduced tumor growth by inducing cellular apoptosis in D54/effluc tumor-bearing mice. Further, the immuno-histological examination of the excised tumor tissue confirmed the presence of apoptotic cells as well as caspase-3 activation. CONCLUSION: Collectively, AP1-ELP-KLAK effectively induced cellular apoptosis of glioblastoma cells and non-invasive imaging provides a window for real-time monitoring of anti-tumor effect with the provision of improving therapeutic efficacy in a glioblastoma mice model.

Anticancer nanocage platforms for combined immunotherapy designed to harness immune checkpoints and deliver anticancer drugs.

The interaction of programmed cell death 1 ligand 1 (PD-L1) with its receptor, programmed cell death 1 (PD-1), inhibits T cell responses. Monoclonal antibodies that block this interaction have been shown effective as immunotherapy. However, only a subset of cancers exhibits a durable response to PD-1/PD-L1 blockade. Moreover, antibody-based immune checkpoint blockade is costly and is occasionally accompanied by systemic side effects. To overcome these limitations of antibody-based immune checkpoint blockade, an immune checkpoint-blocking ferritin nanocage displaying 24 PD-L1 binding peptides (PD-L1pep1) on its surface was designed and constructed. These ferritin nanocages displaying PD-L1pep1 (PpNF) specifically bind to PD-L1 expressed on cancer cells or to purified PD-L1 with a ~30 nM binding affinity. The addition of PpNF to co-cultures of T cells and cancer cells inhibited PD-1/PD-L1 interactions and restored T cell activities. In a mouse model of syngeneic colon cancer, PpNF specifically targeted tumors and showed antitumor activity. Moreover, PpNF nanocages encapsulating the chemotherapeutic drug doxorubicin had more potent antitumor activity than a monoclonal antibody against PD-L1. These results demonstrate that ferritin nanocages displaying surface PD-L1pep1 can be efficiently applied for immunotherapy, especially when encapsulating small chemotherapeutic drugs. These nanocages may have promise as an immunotherapeutic nanomedicine against various solid tumors.

Development of a Noninvasive KIM-1-Based Live-Imaging Technique in the Context of a Drug-Induced Kidney-Injury Mouse Model.

The development of reliable methods to diagnose acute kidney injury is essential to allow the adoption of early therapeutic interventions and evaluate their effectiveness. Based on the fact that kidney injury molecule-1 (KIM-1) expression levels in kidneys are markedly upregulated early after a damage event, here we developed a noninvasive KIM-1-based molecular imaging technique to detect kidney injury. First, we took advantage of a phage-display platform to select small peptides demonstrating a specific high binding affinity to KIM-1. The promising candidate was conjugated with fluorescent probes, and its imaging potential was validated in vitro and in vivo. This peptide, with the sequence CNRRRA, not only showed a high imaging potential in vitro, allowing a strong detection of KIM-1 expressing cells by microscopy and flow cytometry but also generated a strong kidney-specific signal in live-imaging in vivo experiments in the context of a drug-induced kidney-injury mouse model. Our data overall suggest that the CNRRRA peptide is a promising probe to use in the context of in vivo imaging for the detection of KIM-1 overexpression in damaged kidneys.

RGS2-mediated translational control mediates cancer cell dormancy and tumor relapse.

Slow-cycling/dormant cancer cells (SCCs) have pivotal roles in driving cancer relapse and drug resistance. A mechanistic explanation for cancer cell dormancy and therapeutic strategies targeting SCCs are necessary to improve patient prognosis, but are limited because of technical challenges to obtaining SCCs. Here, by applying proliferation-sensitive dyes and chemotherapeutics to non-small cell lung cancer (NSCLC) cell lines and patient-derived xenografts, we identified a distinct SCC subpopulation that resembled SCCs in patient tumors. These SCCs displayed major dormancy-like phenotypes and high survival capacity under hostile microenvironments through transcriptional upregulation of regulator of G protein signaling 2 (RGS2). Database analysis revealed RGS2 as a biomarker of retarded proliferation and poor prognosis in NSCLC. We showed that RGS2 caused prolonged translational arrest in SCCs through persistent eukaryotic initiation factor 2 (eIF2α) phosphorylation via proteasome-mediated degradation of activating transcription factor 4 (ATF4). Translational activation through RGS2 antagonism or the use of phosphodiesterase 5 inhibitors, including sildenafil (Viagra), promoted ER stress-induced apoptosis in SCCs in vitro and in vivo under stressed conditions, such as those induced by chemotherapy. Our results suggest that a low-dose chemotherapy and translation-instigating pharmacological intervention in combination is an effective strategy to prevent tumor progression in NSCLC patients after rigorous chemotherapy.

Tumor-targeting peptide conjugated pH-responsive micelles as a potential drug carrier for cancer therapy.

Herein, we prepared tumor-targeting peptide (AP peptide; CRKRLDRN) conjugated pH-responsive polymeric micelles (pH-PMs) in cancer therapy by active and pH-responsive tumor targeting delivery systems, simultaneously. The active tumor targeting and tumoral pH-responsive polymeric micelles were prepared by mixing AP peptide conjugated PEG-poly(d,l-lactic acid) block copolymer (AP-PEG-PLA) into the pH-responsive micelles of methyl ether poly(ethylene glycol) (MPEG)-poly(beta-amino ester) (PAE) block copolymer (MPEG-PAE). These mixed amphiphilic block copolymers were self-assembled to form stable AP peptide-conjugated and pH-responsive AP-PEG-PLA/MPEG-PAE micelles (AP-pH-PMs) with an average size of 150 nm. The AP-pH-PMs containing 10 wt % of AP-PEG-PLA showed a sharp pH-dependent micellization/demicellization transition at the tumoral acid pH. Also, they presented the pH-dependent drug release profile at the acidic pH of 6.4. The fluorescence dye, TRITC, encapsulated AP-pH-PMs (TRITC-AP-pH-PMs) presented the higher tumor-specific targeting ability in vitro cancer cell culture system and in vivo tumor-bearing mice, compared to control pH-responsive micelles of MPEG-PAE. For the cancer therapy, the anticancer drug, doxorubicin (DOX), was efficiently encapsulated into the AP-pH-PMs (DOX-AP-pH-PMs) with a higher loading efficiency. DOX-AP-pH-PMs efficiently deliver anticancer drugs in MDA-MB231 human breast tumor-bearing mice, resulted in excellent anticancer therapeutic efficacy, compared to free DOX and DOX encapsulated MEG-PAE micelles, indicating the excellent tumor targeting ability of AP-pH-PMs. Therefore, these tumor-targeting peptide-conjugated and pH-responsive polymeric micelles have great potential application in cancer therapy.

Polymorphisms in apoptosis-related genes and survival of patients with early-stage non-small-cell lung cancer.

PURPOSE: This study was conducted to determine the association between single-nucleotide polymorphisms (SNPs) in apoptosis-related genes and survival outcomes of patients with early-stage non-small-cell lung cancer (NSCLC). METHODS: Three hundred ten consecutive patients with surgically resected NSCLC were enrolled. Twenty-five SNPs in 17 apoptosis-related genes were genotyped by a sequenome mass spectrometry-based genotyping assay. The genotype associations with overall survival (OS) and disease-free survival (DFS) were analyzed. RESULTS: Three SNPs (TNFRSF10B rs1047266, TNFRSF1A rs4149570, and PPP1R13L rs1005165) were significantly associated with survival outcomes on multivariate analysis. When the three SNPs were combined, OS and DFS were decreased as the number of bad genotypes increased (P (trend) for OS and DFS = 7 × 10(-5) and 1 × 10(-4), respectively). Patients with one bad genotype, and patients with two or three bad genotypes had significantly worse OS and DFS compared with those with no bad genotypes [adjusted hazard ratio (aHR) for OS = 2.27, 95% confidence interval (CI) = 1.22-4.21, P = 0.01, aHR for DFS = 1.74, 95% CI = 1.08-2.81, P = 0.02; aHR for OS = 4.11, 95% CI = 2.03-8.29, P = 8 × 10(-5); and aHR for DFS = 2.89, 95% CI = 1.64-5.11, P = 3 × 10(-4), respectively]. CONCLUSION: Three SNPs in apoptosis-related genes were identified as possible prognostic markers of survival in patients with early-stage NSCLC. The SNPs, and particularly their combined genotypes, can be used to identify patients at high risk for poor disease outcome.

Targeting of Cisplatin-Resistant Melanoma Using a Multivalent Ligand Presenting an Elastin-like Polypeptide.

Acquired drug resistance is a common occurrence and the main cause of melanoma treatment failure. Melanoma cells frequently developed resistance against cisplatin during chemotherapy, and thus, targeting delivery systems have been devised to decrease drug resistance, increase therapeutic efficacy, and reduce side effects. We genetically engineered a macromolecular carrier using the recursive directional ligation method that specifically targets cisplatin-resistant (Cis-R) melanoma. This carrier is composed of an elastin-like polypeptide (ELP) and multiple copies of Cis-R melanoma-targeting ligands (M-peptide). The designed M16E108 contains 16 targeting ligands incorporated within an ELP and has an ideal thermal phase transition at 39 °C. When treated to melanoma cells, M16E108 specifically accumulated in Cis-R B16F10 melanoma cells and accumulated to a lesser extent in parental B16F10 cells. Consistently, M16E108 exhibited efficient homing and longer retention in tumor tissues in Cis-R melanoma-bearing mice than in parental B16F10 melanoma-bearing mice. Thus, M16E108 was found to display considerable potential as a novel agent that specifically targets cisplatin-resistant melanoma.

Effects of molecular weight and structural conformation of multivalent-based elastin-like polypeptides on tumor accumulation and tissue biodistribution.

In order to improve clinical outcomes for novel drug delivery systems, distinct optimization of size, shape, multifunctionality, and site-specificity are of utmost importance. In this study, we designed various multivalent elastin-like polypeptide (ELP)-based tumor-targeting polymers in which multiple copies of IL-4 receptor (IL-4R)-targeting ligand (AP1 peptide) were periodically incorporated into the ELP polymer backbone to enhance the affinity and avidity towards tumor cells expressing high levels of IL-4R. Several ELPs with different molecular sizes and structures ranging from unimer to micelle-forming polymers were evaluated for their tumor accumulation as well as in vivo bio-distribution patterns. Different percentages of cell binding and uptake were detected corresponding to polymer size, number of targeting peptides, or unimer versus micelle structure. As compared to low molecular weight polypeptides, high molecular weight AP1-ELP showed superior binding activity with faster entry and efficient processing in the IL-4R-dependent endocytic pathway. In addition, in vivo studies revealed that the high molecular weight micelle-forming AP1-ELPs (A86 and A100) displayed better tumor penetration and extensive retention in tumor tissue along with reduced non-specific accumulation in vital organs, when compared to low molecular weight non-micelle forming AP1-ELPs. It is suggested that the superior binding activities shown by A86 and A100 may depend on the multiple presentation of ligands upon transition to a micelle-like structure rather than a larger molecular weight. Thus, this study has significance in elucidating the different patterns underlying unimer and micelle-forming ELP-mediated tumor targeting as well as the in vivo biodistribution.

Polyproline-type helical-structured low-molecular weight heparin (LMWH)-taurocholate conjugate as a new angiogenesis inhibitor.

Although heparin can regulate angiogenesis, tumor growth and metastasis, its clinical application, as well as that of low-molecular heparin (LMWH), for treating cancer are limited because of heparin's anticoagulant activity and risk of hemorrhages. LMWH-taurocholate conjugates (LHT7), which have low anticoagulant activity, were synthesized. The structural property of LHT was evaluated by circular dichroism and the binding affinity of LHT7 to vascular endothelial growth factor 165 (VEGF(165)) was measured by isothermal titration calorimetry. The inhibitory effect of LHT7 on VEGF-mediated KDR (VEGF-receptor 2) phosphorylation in Human umbilical vein endothelial cells was evaluated. The VEGF(165) dependent Matrigel plug assay was performed to verify the antiangiogenic potential of LHT7 on a VEGF(165) inhibitor. Finally, tumor growth inhibition effects of LHT7 on SCC7 and the survival rate of animal models were investigated. Moreover, MDA-MB231 xenograft mouse model was additionally used to confirm the therapeutic effect of LHT7 on human breast cancer cell line. As a result, LHT7 which has 12.7% of anticoagulant activity of the original LMWH showed a peculiar polyproline-type helical structure. LHT7 binds to VEGF strongly and inhibits VEGF dependent KDR phosphorylation. The results of Matrigel plug assay proved LHT7 as a strong antiangiogenic agent inhibiting VEGF(165). Remarkably, LHT7 showed a significant tumor growth inhibition potential on SCC7 with an increased survival rate. LHT7 also delayed tumor growth in MDA-MB231 human breast cancer cell lines.