Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function.

CAR (CARSKNKDC) is a wound-homing peptide that recognises angiogenic neovessels. Here we discover that systemically administered CAR peptide has inherent ability to promote wound healing: wounds close and re-epithelialise faster in CAR-treated male mice. CAR promotes keratinocyte migration in vitro. The heparan sulfate proteoglycan syndecan-4 regulates cell migration and is crucial for wound healing. We report that syndecan-4 expression is restricted to epidermis and blood vessels in mice skin wounds. Syndecan-4 regulates binding and internalisation of CAR peptide and CAR-mediated cytoskeletal remodelling. CAR induces syndecan-4-dependent activation of the small GTPase ARF6, via the guanine nucleotide exchange factor cytohesin-2, and promotes syndecan-4-, ARF6- and Cytohesin-2-mediated keratinocyte migration. Finally, we show that genetic ablation of syndecan-4 in male mice eliminates CAR-induced wound re-epithelialisation following systemic administration. We propose that CAR peptide activates syndecan-4 functions to selectively promote re-epithelialisation. Thus, CAR peptide provides a therapeutic approach to enhance wound healing in mice; systemic, yet target organ- and cell-specific.

Tumor-resident regulatory T cells in pancreatic cancer express the αvß5 integrin as a targetable activation marker.

Pancreatic ductal adenocarcinoma (PDAC) has abundant immunosuppressive regulatory T cells (Tregs), which contribute to a microenvironment resistant to immunotherapy. Here, we report that Tregs in the PDAC tissue, but not those in the spleen, express the αvß5 integrin in addition to neuropilin-1 (NRP-1), which makes them susceptible to the iRGD tumor-penetrating peptide, which targets cells positive for αv integrin- and NRP-1. As a result, long-term treatment of PDAC mice with iRGD leads to tumor-specific depletion of Tregs and improved efficacy of immune checkpoint blockade. αvß5 integrin + Tregs are induced from both naïve CD4 + T cells and natural Tregs upon T cell receptor stimulation, and represent a highly immunosuppressive subpopulation of CCR8 + Tregs. This study identifies the αvß5 integrin as a marker for activated tumor-resident Tregs, which can be targeted to achieve tumor-specific Treg depletion and thereby augment anti-tumor immunity for PDAC therapy.

Reflections on Integrins-Past, Present, and Future: The Albert Lasker Basic Medical Research Award.

This Viewpoint discusses the rapid advances in molecular cell biological approaches over the past 50 years and the many avenues for future advances that have been opened, including direct applications for therapeutic and regenerative medicine.

Molecular ZIP codes in targeted drug delivery.

The term "molecular ZIP (or area) codes" refers to an originally hypothetical system of cell adhesion molecules that would control cell trafficking in the body. Subsequent discovery of the integrins, cadherins, and other cell adhesion molecules confirmed this hypothesis. The recognition system encompassing integrins and their ligands came particularly close to fulfilling the original ZIP code hypothesis, as multiple integrins with closely related specificities mediate cell adhesion by binding to an RGD or related sequence in various extracellular matrix proteins. Diseased tissues have their own molecular addresses that, although not necessarily involved in cell trafficking, can be made use of in targeted drug delivery. This article discusses the molecular basis of ZIP codes and the extensive effort under way to harness them for drug delivery purposes.

iRGD-liposomes enhance tumor delivery and therapeutic efficacy of antisense oligonucleotide drugs against primary prostate cancer and bone metastasis.

Nucleotide-based drugs, such as antisense oligonucleotides (ASOs), have unique advantages in treating human diseases as they provide virtually unlimited ability to target any gene. However, their clinical translation faces many challenges, one of which is poor delivery to the target tissue in vivo. This problem is particularly evident in solid tumors. Here, we functionalized liposomes with a tumor-homing and -penetrating peptide, iRGD, as a carrier of an ASO against androgen receptor (AR) for prostate cancer treatment. The iRGD-liposomes exhibited a high loading efficiency of AR-ASO, and an efficient knockdown of AR gene products was achieved in vitro, including AR splice variants. In vivo, iRGD-liposomes significantly increased AR-ASO accumulation in the tumor tissue and decreased AR expression relative to free ASOs in prostate tumors established as subcutaneous xenografts. Similar results were obtained with intra-tibial xenografts modeling metastasis to bones, the predominant site of metastasis for prostate cancer. In treatment studies, iRGD-liposomes markedly improved the AR-ASO efficacy in suppressing the growth of both subcutaneous xenografts and intra-tibial xenografts. The inhibitory effect on tumor growth was also significantly prolonged by the delivery of the AR-ASO in the iRGD-liposomes. Meanwhile, iRGD-liposomes did not increase ASO accumulation or toxicity in healthy organs. Overall, we provide here a delivery system that can significantly increase ASO accumulation and efficacy in solid tumors. These benefits are achieved without significant side effects, providing a way to increase the antitumor efficacy of ASOs.

Tumor-penetrating therapy for ß5 integrin-rich pancreas cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked desmoplasia and drug resistance due, in part, to poor drug delivery to extravascular tumor tissue. Here, we report that carcinoma-associated fibroblasts (CAFs) induce ß5 integrin expression in tumor cells in a TGF-ß dependent manner, making them an efficient drug delivery target for the tumor-penetrating peptide iRGD. The capacity of iRGD to deliver conjugated and co-injected payloads is markedly suppressed when ß5 integrins are knocked out in the tumor cells. Of note, ß5 integrin knock-out in tumor cells leads to reduced disease burden and prolonged survival of the mice, demonstrating its contribution to PDAC progression. iRGD significantly potentiates co-injected chemotherapy in KPC mice with high ß5 integrin expression and may be a powerful strategy to target an aggressive PDAC subpopulation.

Fusogenic porous silicon nanoparticles as a broad-spectrum immunotherapy against bacterial infections.

Bacterial infections are re-emerging as substantial threats to global health due to the limited selection of antibiotics that are capable of overcoming antibiotic-resistant strains. By deterring such mutations whilst minimizing the need to develop new pathogen-specific antibiotics, immunotherapy offers a broad-spectrum therapeutic solution against bacterial infections. In particular, pathology resulting from excessive immune response (i.e. fibrosis, necrosis, exudation, breath impediment) contributes significantly to negative disease outcome. Herein, we present a nanoparticle that is targeted to activated macrophages and loaded with siRNA against the Irf5 gene. This formulation is able to induce >80% gene silencing in activated macrophages in vivo, and it inhibits the excessive inflammatory response, generating a significantly improved therapeutic outcome in mouse models of bacterial infection. The versatility of the approach is demonstrated using mice with antibiotic-resistant Gram-positive (methicillin-resistant Staphylococcus aureus) and Gram-negative (Pseudomonas aeruginosa) muscle and lung infections, respectively. Effective depletion of the Irf5 gene in macrophages is found to significantly improve the therapeutic outcome of infected mice, regardless of the bacteria strain and type.

Systemic brain tumor delivery of synthetic protein nanoparticles for glioblastoma therapy.

Glioblastoma (GBM), the most aggressive form of brain cancer, has witnessed very little clinical progress over the last decades, in part, due to the absence of effective drug delivery strategies. Intravenous injection is the least invasive drug delivery route to the brain, but has been severely limited by the blood-brain barrier (BBB). Inspired by the capacity of natural proteins and viral particulates to cross the BBB, we engineered a synthetic protein nanoparticle (SPNP) based on polymerized human serum albumin (HSA) equipped with the cell-penetrating peptide iRGD. SPNPs containing siRNA against Signal Transducer and Activation of Transcription 3 factor (STAT3i) result in in vitro and in vivo downregulation of STAT3, a central hub associated with GBM progression. When combined with the standard of care, ionized radiation, STAT3i SPNPs result in tumor regression and long-term survival in 87.5% of GBM-bearing mice and prime the immune system to develop anti-GBM immunological memory.

Immune-mediated ECM depletion improves tumour perfusion and payload delivery.

High extracellular matrix (ECM) content in solid cancers impairs tumour perfusion and thus access of imaging and therapeutic agents. We have devised a new approach to degrade tumour ECM, which improves uptake of circulating compounds. We target the immune-modulating cytokine, tumour necrosis factor alpha (TNFα), to tumours using a newly discovered peptide ligand referred to as CSG. This peptide binds to laminin-nidogen complexes in the ECM of mouse and human carcinomas with little or no peptide detected in normal tissues, and it selectively delivers a recombinant TNFα-CSG fusion protein to tumour ECM in tumour-bearing mice. Intravenously injected TNFα-CSG triggered robust immune cell infiltration in mouse tumours, particularly in the ECM-rich zones. The immune cell influx was accompanied by extensive ECM degradation, reduction in tumour stiffness, dilation of tumour blood vessels, improved perfusion and greater intratumoral uptake of the contrast agents gadoteridol and iron oxide nanoparticles. Suppressed tumour growth and prolonged survival of tumour-bearing mice were observed. These effects were attainable without the usually severe toxic side effects of TNFα.

Securing the Payload, Finding the Cell, and Avoiding the Endosome: Peptide-Targeted, Fusogenic Porous Silicon Nanoparticles for Delivery of siRNA.

Despite the promise of ribonucleic acid interference therapeutics, the delivery of oligonucleotides selectively to diseased tissues in the body, and specifically to the cellular location in the tissues needed to provide optimal therapeutic outcome, remains a significant challenge. Here, key material properties and biological mechanisms for delivery of short interfering RNAs (siRNAs) to effectively silence target-specific cells in vivo are identified. Using porous silicon nanoparticles as the siRNA host, tumor-targeting peptides for selective tissue homing, and fusogenic lipid coatings to induce fusion with the plasma membrane, it is shown that the uptake mechanism can be engineered to be independent of common receptor-mediated endocytosis pathways. Two examples of the potential broad clinical applicability of this concept in a mouse xenograft model of ovarian cancer peritoneal carcinomatosis are provided: silencing the Rev3l subunit of polymerase Pol ζ to impair DNA repair in combination with cisplatin; and reprogramming tumor-associated macrophages into a proinflammatory state.

Peptide-guided nanoparticles for glioblastoma targeting.

Tumor-selective drug conjugates can potentially improve the prognosis for patients affected by glioblastoma (GBM) - the most common and malignant type of brain cancer with no effective cure. Here we evaluated a novel tumor penetrating peptide that targets cell surface p32, LinTT1 (AKRGARSTA), as a GBM targeting ligand for systemically-administered nanoparticles. LinTT1-functionalization increased tumor homing of iron oxide nanoworms (NWs) across a panel of five GBM models ranging from infiltratively-disseminating to angiogenic phenotypes. LinTT1-NWs homed to CD31-positive tumor blood vessels, including to transdifferentiated endothelial cells, and showed co-localization with tumor macrophages and lymphatic vessels. LinTT1 functionalization also resulted in increased GBM delivery of other types of systemically-administered nanoparticles: silver nanoparticles and albumin-paclitaxel nanoparticles. Finally, LinTT1-guided proapoptotic NWs exerted strong anti-glioma activity in two models of GBM, including doubling the lifespan of the mice in an aggressive orthotopic stem cell-like GBM that recapitulates the histological hallmarks of human GBM. Our study suggests that LinTT1 targeting strategy can be used to increase GBM uptake of systemic nanoparticles for improved imaging and therapy.

Tumor-Targeting, MicroRNA-Silencing Porous Silicon Nanoparticles for Ovarian Cancer Therapy.

Silencing of aberrantly expressed microRNAs (miRNAs or miRs) has emerged as one of the strategies for molecular targeted cancer therapeutics. In particular, miR-21 is an oncogenic miRNA overexpressed in many tumors, including ovarian cancer. To achieve efficient administration of anti-miR therapeutics, delivery systems are needed that can ensure local accumulation in the tumor environment, low systemic toxicity, and reduced adverse side effects. In order to develop an improved anti-miR therapeutic agent for the treatment of ovarian cancer, a nanoformulation is engineered that leverages biodegradable porous silicon nanoparticles (pSiNPs) encapsulating an anti-miR-21 locked nucleic acid payload and displaying a tumor-homing peptide for targeted distribution. Targeting efficacy, miR-21 silencing, and anticancer activity are optimized in vitro on a panel of ovarian cancer cell lines, and a formulation of anti-miR-21 in a pSiNP displaying the targeting peptide CGKRK is identified for in vivo evaluation. When this nanoparticulate agent is delivered to mice bearing tumor xenografts, a substantial inhibition of tumor growth is achieved through silencing of miR-21. This study presents the first successful application of tumor-targeted anti-miR porous silicon nanoparticles for the treatment of ovarian cancer in a mouse xenograft model.

Tumor-specific macrophage targeting through recognition of retinoid X receptor beta.

Macrophages play important and diverse roles during cancer progression. However, cancer therapies based on macrophage modulation are lacking in tools that can recognize and deliver therapeutic payloads to macrophages in a tumor-specific manner. As a result, treatments tend to interfere with normal macrophage functions in healthy organs. We previously identified a macrophage-binding peptide, termed CRV. Here, we show that upon systemic administration into tumor-bearing mice, CRV selectively homes to tumors, extravasates, and preferentially binds to macrophages within. CRV exhibits a higher affinity for tumor macrophages than for other cells in tumors or for other macrophage types elsewhere in the body. We further identified and validated retinoid X receptor beta (RXRB) as the CRV receptor. Intriguingly, although it is known as a nuclear receptor, RXRB shows a prominent cell surface localization that is largely restricted to tumor macrophages. Systemic administration of anti-RXRB antibodies also results in tumor-selective binding to macrophages similar to CRV. Lastly, we demonstrate the ability of CRV to improve the delivery of nano-carriers into solid tumors and macrophages within. In summary, we describe here a novel cell surface marker and targeting tools for tumor macrophages that may aid in future development of macrophage-modulatory cancer therapies.

Generation of a multi-functional, target organ-specific, anti-fibrotic molecule by molecular engineering of the extracellular matrix protein, decorin.

Extracellular matrix (ECM) molecules play important roles in regulating processes such as cell proliferation, migration, differentiation and survival. Decorin is a proteoglycan that binds to ('decorates') collagen fibrils in the ECM. Decorin also interacts with many growth factors and their receptors, the most notable of these interactions being its inhibitory activity on TGF-ß, the growth factor responsible for fibrosis formation. We have generated a recombinant, multi-functional, fusion-protein consisting of decorin as a therapeutic domain and a vascular homing and cell-penetrating peptide as a targeting vehicle. This recombinant decorin (CAR-DCN) accumulates at the sites of the targeted disease at higher levels and, as a result, has substantially enhanced biological activity over native decorin. CAR-DCN is an example of how molecular engineering can give a compound the ability to seek out sites of disease and enhance its therapeutic potential. CAR-DCN will hopefully be used to treat severe human diseases. LINKED ARTICLES: This article is part of a themed section on Translating the Matrix. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.1/issuetoc.

Tracking the Fate of Porous Silicon Nanoparticles Delivering a Peptide Payload by Intrinsic Photoluminescence Lifetime.

A nanoparticle system for systemic delivery of therapeutics is described, which incorporates a means of tracking the fate of the nanocarrier and its residual drug payload in vivo by photoluminescence (PL). Porous silicon nanoparticles (PSiNPs) containing the proapoptotic antimicrobial peptide payload, D [KLAKLAK]2 , are monitored by measurement of the intrinsic PL intensity and the PL lifetime of the nanoparticles. The PL lifetime of the PSiNPs is on the order of microseconds, substantially longer than the nanosecond lifetimes typically exhibited by conventional fluorescent tags or by autofluorescence from cells and tissues; thus, emission from the nanoparticles is readily discerned in the time-resolved PL spectrum. It is found that the luminescence lifetime of the PSiNP host decreases as the nanoparticle dissolves in phosphate-buffered saline solution (37 °C), and this correlates with the extent of release of the peptide payload. The time-resolved PL measurement allows tracking of the in vivo fate of PSiNPs injected (via tail vein) into mice. Clearance of the nanoparticles through the liver, kidneys, and lungs of the animals is observed. The luminescence lifetime of the PSiNPs decreases with increasing residence time in the mice, providing a measure of half-life for degradation of the drug nanocarriers.

Antibiotic-loaded nanoparticles targeted to the site of infection enhance antibacterial efficacy.

Bacterial resistance to antibiotics has made it necessary to resort to antibiotics that have considerable toxicities. Here, we show that the cyclic 9-amino acid peptide CARGGLKSC (CARG), identified via phage display on Staphylococcus aureus (S. aureus) bacteria and through in vivo screening in mice with S. aureus-induced lung infections, increases the antibacterial activity of CARG-conjugated vancomycin-loaded nanoparticles in S. aureus-infected tissues and reduces the needed overall systemic dose, minimizing side effects. CARG binds specifically to S. aureus bacteria but not Pseudomonas bacteria in vitro, selectively accumulates in S. aureus-infected lungs and skin of mice but not in non-infected tissue and Pseudomonas-infected tissue, and significantly enhances the accumulation of intravenously injected vancomycin-loaded porous silicon nanoparticles bearing the peptide in S. aureus-infected mouse lung tissue. The targeted nanoparticles more effectively suppress staphylococcal infections in vivo relative to equivalent doses of untargeted vancomycin nanoparticles or of free vancomycin. The therapeutic delivery of antibiotic-carrying nanoparticles bearing peptides targeting infected tissue may help combat difficult-to-treat infections.

Immunogene therapy with fusogenic nanoparticles modulates macrophage response to Staphylococcus aureus.

The incidence of adverse effects and pathogen resistance encountered with small molecule antibiotics is increasing. As such, there is mounting focus on immunogene therapy to augment the immune system's response to infection and accelerate healing. A major obstacle to in vivo gene delivery is that the primary uptake pathway, cellular endocytosis, results in extracellular excretion and lysosomal degradation of genetic material. Here we show a nanosystem that bypasses endocytosis and achieves potent gene knockdown efficacy. Porous silicon nanoparticles containing an outer sheath of homing peptides and fusogenic liposome selectively target macrophages and directly introduce an oligonucleotide payload into the cytosol. Highly effective knockdown of the proinflammatory macrophage marker IRF5 enhances the clearance capability of macrophages and improves survival in a mouse model of Staphyloccocus aureus pneumonia.

Publisher Correction: Identification of a peptide recognizing cerebrovascular changes in mouse models of Alzheimer's disease.

The original version of the Supplementary Information associated with this Article inadvertently omitted Supplementary Table 1. The HTML has now been updated to include a corrected version of the Supplementary Information.

Selective Targeting of a Novel Vasodilator to the Uterine Vasculature to Treat Impaired Uteroplacental Perfusion in Pregnancy.

Fetal growth restriction (FGR) in pregnancy is commonly caused by impaired uteroplacental blood flow. Vasodilators enhance uteroplacental perfusion and fetal growth in humans and animal models; however, detrimental maternal and fetal side effects have been reported. We hypothesised that targeted uteroplacental delivery of a vasodilator would enhance drug efficacy and reduce the risks associated with drug administration in pregnancy. Phage screening identified novel peptides that selectively accumulated in the uteroplacental vasculature of pregnant mice. Following intravenous injection, the synthetic peptide CNKGLRNK selectively bound to the endothelium of the uterine spiral arteries and placental labyrinth in vivo; CNKGLRNK-decorated liposomes also selectively bound to these regions. The nitric oxide donor 2-[[4-[(nitrooxy)methyl]benzoyl]thio]-benzoic acid methyl ester (SE175) induced significant relaxation of mouse uterine arteries and human placental arteries in vitro; thus, SE175 was encapsulated into these targeted liposomes and administered to healthy pregnant C57BL/6J mice or endothelial nitric oxide synthase knockout (eNOS-/-) mice, which exhibit impaired uteroplacental blood flow and FGR. Liposomes containing SE175 (0.44mg/kg) or PBS were administered on embryonic (E) days 11.5, 13.5, 15.5 and 17.5; fetal and placental weights were recorded at term and compared to mice injected with free PBS or SE175. Targeted uteroplacental delivery of SE175 had no effect on fetal weight in C57BL/6J mice, but significantly increased fetal weight and mean spiral artery diameter, and decreased placental weight, indicative of improved placental efficiency, in eNOS-/- mice; free SE175 had no effect on fetal weight or spiral artery diameter. Targeted, but not free SE175 also significantly reduced placental expression of 4-hydroxynonenal, cyclooxygenase-1 and cyclooxygenase-2, indicating a reduction in placental oxidative stress. These data suggest that exploiting vascular targeting peptides to selectively deliver SE175 to the uteroplacental vasculature may represent a novel treatment for FGR resulting from impaired uteroplacental perfusion.