ESCPE-1 mediates retrograde endosomal sorting of the SARS-CoV-2 host factor Neuropilin-1.

Endosomal sorting maintains cellular homeostasis by recycling transmembrane proteins and associated proteins and lipids (termed "cargoes") from the endosomal network to multiple subcellular destinations, including retrograde traffic to the trans-Golgi network (TGN). Viral and bacterial pathogens subvert retrograde trafficking machinery to facilitate infectivity. Here, we develop a proteomic screen to identify retrograde cargo proteins of the endosomal SNX-BAR sorting complex promoting exit 1 (ESCPE-1). Using this methodology, we identify Neuropilin-1 (NRP1), a recently characterized host factor for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, as a cargo directly bound and trafficked by ESCPE-1. ESCPE-1 mediates retrograde trafficking of engineered nanoparticles functionalized with the NRP1-interacting peptide of the SARS-CoV-2 spike (S) protein. CRISPR-Cas9 deletion of ESCPE-1 subunits reduces SARS-CoV-2 infection levels in cell culture. ESCPE-1 sorting of NRP1 may therefore play a role in the intracellular membrane trafficking of NRP1-interacting viruses such as SARS-CoV-2.

Preclinical Validation of Tumor-Penetrating and Interfering Peptides against Chronic Lymphocytic Leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults. The disease is characterized by the accumulation of tumoral B cells resulting from a defect of apoptosis. We have in vitro and in vivo preclinically validated a tumor-penetrating peptide (named TT1) coupled to an interfering peptide (IP) that dissociates the interaction between the serine/threonine protein phosphatase 2A (PP2A) from its physiological inhibitor, the oncoprotein SET. This TT1-IP peptide has an antitumoral effect on CLL, as shown by the increased survival of mice bearing xenograft models of CLL, compared to control mice. The peptide did not show toxicity, as indicated by the mouse body weight and the biochemical parameters, such as renal and hepatic enzymes. In addition, the peptide-induced apoptosis in vitro of primary tumoral B cells isolated from CLL patients but not of those isolated from healthy patients. Finally, the peptide had approximately 5 h half-life in human serum and showed pharmacokinetic parameters compatible with clinical development as a therapeutic peptide against CLL.

Novel Anthracycline Utorubicin for Cancer Therapy.

Novel anticancer compounds and their precision delivery systems are actively developed to create potent and well-tolerated anticancer therapeutics. Here, we report the synthesis of a novel anthracycline, Utorubicin (UTO), and its preclinical development as an anticancer payload for nanocarriers. Free UTO was significantly more toxic to cultured tumor cell lines than the clinically used anthracycline, doxorubicin. Nanoformulated UTO, encapsulated in polymeric nanovesicles (polymersomes, PS), reduced the viability of cultured malignant cells and this effect was potentiated by functionalization with a tumor-penetrating peptide (TPP). Systemic peptide-guided PS showed preferential accumulation in triple-negative breast tumor xenografts implanted in mice. At the same systemic UTO dose, the highest UTO accumulation in tumor tissue was seen for the TPP-targeted PS, followed by nontargeted PS, and free doxorubicin. Our study suggests potential applications for UTO in the treatment of malignant diseases and encourages further preclinical and clinical studies on UTO as a nanocarrier payload for precision cancer therapy.

Targeting Pro-Tumoral Macrophages in Early Primary and Metastatic Breast Tumors with the CD206-Binding mUNO Peptide.

M2-like tumor-associated macrophages (M2 TAMs) play important roles in the resistance of tumors to immunotherapies. Selective depletion or reprogramming of M2 TAMs may sensitize the nonresponsive tumors for immune-mediated eradication. However, precision delivery of payloads to M2 TAMs, while sparing healthy tissues, has remained an unresolved challenge. Here, we studied the application of a short linear peptide (CSPGAK, "mUNO") for the delivery of molecular and nanoscale cargoes in M2 TAMs in vitro and the relevance of the peptide for in vivo targeting of early-stage primary breast tumors and metastatic lung foci. First, we performed in silico modeling and found that mUNO interacts with mouse CD206 via a binding site between lectin domains CTLD1 and CTLD2, the same site previously demonstrated to be involved in mUNO binding to human CD206. Second, we showed that cultured M2 macrophages take up fluorescein-labeled (FAM) polymersomes conjugated with mUNO using the sulfhydryl group of its N-terminal cysteine. Pulse/chase studies of FAM-mUNO in M2 macrophages suggested that the peptide avoided lysosomal entrapment and escaped from early endosomes. Third, our in vivo studies with FAM-mUNO demonstrated that intraperitoneal administration results in better pharmacokinetics and higher blood bioavailability than can be achieved with intravenous administration. Intraperitoneal FAM-mUNO, but not FAM-control, showed a robust accumulation in M2-skewed macrophages in mouse models of early primary breast tumor and lung metastasis. This targeting was specific, as no uptake was observed in nonmalignant control organs, including the liver, or other cell types in the tumor, including M1 macrophages. Collectively, our studies support the application of the CD206-binding mUNO peptide for delivery of molecular and nanoscale cargoes to M2 macrophages and manifest the relevance of this mode of targeting primary and metastatic breast tumors.

iRGD-guided tamoxifen polymersomes inhibit estrogen receptor transcriptional activity and decrease the number of breast cancer cells with self-renewing capacity.

BACKGROUND: Tamoxifen (Tam) is the most frequent treatment for estrogen receptor (ER) positive breast cancer. We recently showed that fibronectin (FN) leads to Tam resistance and selection of breast cancer stem cells. With the aim of developing a nanoformulation that would simultaneously tackle ER and FN/ß1 integrin interactions, we designed polyethylene glycol-polycaprolactone polymersomes polymersomes (PS) that carry Tam and are functionalized with the tumor-penetrating iRGD peptide (iRGD-PS-Tam). RESULTS: Polyethylene glycol-polycaprolactone PS were assembled and loaded with Tam using the hydration film method. The loading of encapsulated Tam, measured by UPLC, was 2.4 ± 0.5 mol Tam/mol polymer. Physicochemical characterization of the PS demonstrated that iRGD functionalization had no effect on morphology, and a minimal effect on the PS size and polydispersity (176 nm and Pdi 0.37 for iRGD-TAM-PS and 171 nm and Pdi 0.36 for TAM-PS). iRGD-PS-Tam were taken up by ER+ breast carcinoma cells in 2D-culture and exhibited increased penetration of 3D-spheroids. Treatment with iRGD-PS-Tam inhibited proliferation and sensitized cells cultured on FN to Tam. Mechanistically, treatment with iRGD-PS-Tam resulted in inhibition ER transcriptional activity as evaluated by a luciferase reporter assay. iRGD-PS-Tam reduced the number of cells with self-renewing capacity, a characteristic of breast cancer stem cells. In vivo, systemic iRGD-PS-Tam showed selective accumulation at the tumor site. CONCLUSIONS: Our study suggests iRGD-guided delivery of PS-Tam as a potential novel therapeutic strategy for the management of breast tumors that express high levels of FN. Future studies in pre-clinical in vivo models are warranted.

Peritoneal Carcinomatosis Targeting with Tumor Homing Peptides.

Over recent decades multiple therapeutic approaches have been explored for improved management of peritoneally disseminated malignancies-a grim condition known as peritoneal carcinomatosis (PC). Intraperitoneal (IP) administration can be used to achieve elevated local concentration and extended half-life of the drugs in the peritoneal cavity to improve their anticancer efficacy. However, IP-administered chemotherapeutics have a short residence time in the IP space, and are not tumor selective. An increasing body of work suggests that functionalization of drugs and nanoparticles with targeting peptides increases their peritoneal retention and provides a robust and specific tumor binding and penetration that translates into improved therapeutic response. Here we review the progress in affinity targeting of intraperitoneal anticancer compounds, imaging agents and nanoparticles with tumor-homing peptides. We review classes of tumor-homing peptides relevant for PC targeting, payloads for peptide-guided precision delivery, applications for targeted compounds, and the effects of nanoformulation of drugs and imaging agents on affinity-based tumor delivery.

Hyaluronan-binding peptide for targeting peritoneal carcinomatosis.

Peritoneal carcinomatosis results from dissemination of solid tumors in the peritoneal cavity, and is a common site of metastasis in patients with carcinomas of gastrointestinal or gynecological origin. Peritoneal carcinomatosis treatment is challenging as poorly vascularized, disseminated peritoneal micro-tumors are shielded from systemic anticancer drugs and drive tumor regrowth. Here, we describe the identification and validation of a tumor homing peptide CKRDLSRRC (IP3), which upon intraperitoneal administration delivers payloads to peritoneal metastases. IP3 peptide was identified by in vivo phage display on a mouse model of peritoneal carcinomatosis of gastric origin (MKN-45P), using high-throughput sequencing of the peptide-encoding region of phage genome as a readout. The IP3 peptide contains a hyaluronan-binding motif, and fluorescein-labeled IP3 peptide bound to immobilized hyaluronan in vitro. After intraperitoneal administration in mice bearing peritoneal metastases of gastric and colon origin, IP3 peptide homed robustly to macrophage-rich regions in peritoneal tumors, including poorly vascularized micro-tumors. Finally, we show that IP3 functionalization conferred silver nanoparticles the ability to home to peritoneal tumors of gastric and colonic origin, suggesting that it could facilitate targeted delivery of nanoscale payloads to peritoneal tumors. Collectively, our study suggests that the IP3 peptide has potential applications for targeting drugs, nanoparticles, and imaging agents to peritoneal tumors.

Biocompatible, multifunctional, and well-defined OEG-based dendritic platforms for biomedical applications.

Given the growing importance of drug and gene delivery systems, imaging agents, biosensors, and theranostics, there is a need to develop new multifunctional and biocompatible platforms. Here we synthesized and fully characterized a family of novel multifunctional and completely monodisperse dendritic platforms. Our synthetic methodology, based on compatible protecting groups and the attachment of monodisperse triethylene glycol units, allows the control of the generation and differentiation of terminal groups, thus giving rise to multifunctional and perfectly-defined products. A family of dendrons was synthesized and four distinct dendritic structures were chosen from the family in order to determine the effect of the generation and surface groups on their biocompatibility. The stability in serum, cytotoxicity, and hemocompatibility of these products were studied. Our results indicate that these non-toxic, hemocompatible, non-immunogenic, stable and versatile scaffolds may be very interesting candidates for biomedical applications.

Targeting of Tomato Bushy Stunt Virus with a Genetically Fused C-End Rule Peptide.

Homing peptides are widely used to improve the delivery of drugs, imaging agents, and nanoparticles (NPs) to their target sites. Plant virus-based particles represent an emerging class of structurally diverse nanocarriers that are biocompatible, biodegradable, safe, and cost-effective. Similar to synthetic NPs, these particles can be loaded with imaging agents and/or drugs and functionalized with affinity ligands for targeted delivery. Here we report the development of a peptide-guided Tomato Bushy Stunt Virus (TBSV)-based nanocarrier platform for affinity targeting with the C-terminal C-end rule (CendR) peptide, RPARPAR (RPAR). Flow cytometry and confocal microscopy demonstrated that the TBSV-RPAR NPs bind specifically to and internalize in cells positive for the peptide receptor neuropilin-1 (NRP-1). TBSV-RPAR particles loaded with a widely used anticancer anthracycline, doxorubicin, showed selective cytotoxicity on NRP-1-expressing cells. Following systemic administration in mice, RPAR functionalization conferred TBSV particles the ability to accumulate in the lung tissue. Collectively, these studies show the feasibility of the CendR-targeted TBSV platform for the precision delivery of payloads.

Paclitaxel-Loaded Cationic Fluid Lipid Nanodiscs and Liposomes with Brush-Conformation PEG Chains Penetrate Breast Tumors and Trigger Caspase-3 Activation.

Novel approaches are required to address the urgent need to develop lipid-based carriers of paclitaxel (PTX) and other hydrophobic drugs for cancer chemotherapy. Carriers based on cationic liposomes (CLs) with fluid (i.e., chain-melted) membranes (e.g., EndoTAG-1) have shown promise in preclinical and late-stage clinical studies. Recent work found that the addition of a cone-shaped poly(ethylene glycol)-lipid (PEG-lipid) to PTX-loaded CLs (CLsPTX) promotes a transition to sterically stabilized, higher-curvature (smaller) nanoparticles consisting of a mixture of PEGylated CLsPTX and PTX-containing fluid lipid nanodiscs (nanodiscsPTX). These CLsPTX and nanodiscsPTX show significantly improved uptake and cytotoxicity in cultured human cancer cells at PEG coverage in the brush regime (10 mol % PEG-lipid). Here, we studied the PTX loading, in vivo circulation half-life, and biodistribution of systemically administered CLsPTX and nanodiscsPTX and assessed their ability to induce apoptosis in triple-negative breast-cancer-bearing immunocompetent mice. We focused on fluid rather than solid lipid nanodiscs because of the significantly higher solubility of PTX in fluid membranes. At 5 and 10 mol % of a PEG-lipid (PEG5K-lipid, molecular weight of PEG 5000 g/mol), the mixture of PEGylated CLsPTX and nanodiscsPTX was able to incorporate up to 2.5 mol % PTX without crystallization for at least 20 h. Remarkably, compared to preparations containing 2 and 5 mol % PEG5K-lipid (with the PEG chains in the mushroom regime), the particles at 10 mol % (with PEG chains in the brush regime) showed significantly higher blood half-life, tumor penetration, and proapoptotic activity. Our study suggests that increasing the PEG coverage of CL-based drug nanoformulations can improve their pharmacokinetics and therapeutic efficacy.

Bi-Functional Peptides as a New Therapeutic Tool for Hepatocellular Carcinoma.

BACKGROUND: The interfering peptides that block protein-protein interactions have been receiving increasing attention as potential therapeutic tools. METHODS: We measured the internalization and biological effect of four bi-functional tumor-penetrating and interfering peptides into primary hepatocytes isolated from three non-malignant and 11 hepatocellular carcinomas. RESULTS: These peptides are internalized in malignant hepatocytes but not in non-malignant cells. Furthermore, the degree of peptide internalization correlated with receptor expression level and tumor aggressiveness levels. Importantly, penetration of the peptides iRGD-IP, LinTT1-IP, TT1-IP, and RPARPAR-IP induced apoptosis of the malignant hepatocytes without effect on non-malignant cells. CONCLUSION: Receptor expression levels correlated with the level of peptide internalization and aggressiveness of the tumor. This study highlights the potential to exploit the expression of tumor-penetrating peptide receptors as a predictive marker of liver tumor aggressiveness. These bi-functional peptides could be developed for personalized tumor treatment.

Novel Anthracycline Utorubicin for Cancer Therapy.

Novel anticancer compounds and their precision delivery systems are actively developed to create potent and well-tolerated anticancer therapeutics. Here, we report the synthesis of a novel anthracycline, Utorubicin (UTO), and its preclinical development as an anticancer payload for nanocarriers. Free UTO was significantly more toxic to cultured tumor cell lines than the clinically used anthracycline, doxorubicin. Nanoformulated UTO, encapsulated in polymeric nanovesicles (polymersomes, PS), reduced the viability of cultured malignant cells and this effect was potentiated by functionalization with a tumor-penetrating peptide (TPP). Systemic peptide-guided PS showed preferential accumulation in triple-negative breast tumor xenografts implanted in mice. At the same systemic UTO dose, the highest UTO accumulation in tumor tissue was seen for the TPP-targeted PS, followed by nontargeted PS, and free doxorubicin. Our study suggests potential applications for UTO in the treatment of malignant diseases and encourages further preclinical and clinical studies on UTO as a nanocarrier payload for precision cancer therapy.

Homing Peptide-Based Targeting of Tenascin-C and Fibronectin in Endometriosis.

The current diagnostic and therapeutic strategies for endometriosis are limited. Although endometriosis is a benign condition, some of its traits, such as increased cell invasion, migration, tissue inflammation, and angiogenesis are similar to cancer. Here we explored the application of homing peptides for precision delivery of diagnostic and therapeutic compounds to endometriotic lesions. First, we audited a panel of peptide phages for the binding to the cultured immortalized endometriotic epithelial 12Z and eutopic stromal HESC cell lines. The bacteriophages displaying PL1 peptide that engages with angiogenic extracellular matrix overexpressed in solid tumors showed the strongest binding to both cell lines. The receptors of PL1 peptide, tenascin C domain C (TNC-C) and fibronectin Extra Domain-B (Fn-EDB), were expressed in both cells. Silver nanoparticles functionalized with synthetic PL1 peptide showed specific internalization in 12Z and HESC cells. Treatment with PL1-nanoparticles loaded with the potent antimitotic drug monomethyl auristatin E decreased the viability of endometriotic cells in 2D and 3D cultures. Finally, PL1-nanoparticless bound to the cryosections of clinical peritoneal endometriotic lesions in the areas positive for TNC-C and Fn-EDB immunoreactivities and not to sections of normal endometrium. Our findings suggest potential applications for PL1-guided nanoparticles in precision diagnosis and therapy of endometriosis.

Cationic Liposomes as Vectors for Nucleic Acid and Hydrophobic Drug Therapeutics.

Cationic liposomes (CLs) are effective carriers of a variety of therapeutics. Their applications as vectors of nucleic acids (NAs), from long DNA and mRNA to short interfering RNA (siRNA), have been pursued for decades to realize the promise of gene therapy, with approvals of the siRNA therapeutic patisiran and two mRNA vaccines against COVID-19 as recent milestones. The long-term goal of developing optimized CL-based NA carriers for a broad range of medical applications requires a comprehensive understanding of the structure of these vectors and their interactions with cell membranes and components that lead to the release and activity of the NAs within the cell. Structure-activity relationships of lipids for CL-based NA and drug delivery must take into account that these lipids act not individually but as components of an assembly of many molecules. This review summarizes our current understanding of how the choice of the constituting lipids governs the structure of their CL-NA self-assemblies, which constitute distinct liquid crystalline phases, and the relation of these structures to their efficacy for delivery. In addition, we review progress toward CL-NA nanoparticles for targeted NA delivery in vivo and close with an outlook on CL-based carriers of hydrophobic drugs, which may eventually lead to combination therapies with NAs and drugs for cancer and other diseases.

Neuropilin-1 is a host factor for SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), uses the viral spike (S) protein for host cell attachment and entry. The host protease furin cleaves the full-length precursor S glycoprotein into two associated polypeptides: S1 and S2. Cleavage of S generates a polybasic Arg-Arg-Ala-Arg carboxyl-terminal sequence on S1, which conforms to a C-end rule (CendR) motif that binds to cell surface neuropilin-1 (NRP1) and NRP2 receptors. We used x-ray crystallography and biochemical approaches to show that the S1 CendR motif directly bound NRP1. Blocking this interaction by RNA interference or selective inhibitors reduced SARS-CoV-2 entry and infectivity in cell culture. NRP1 thus serves as a host factor for SARS-CoV-2 infection and may potentially provide a therapeutic target for COVID-19.

Phage-Display-Derived Peptide Binds to Human CD206 and Modeling Reveals a New Binding Site on the Receptor.

We recently identified a tumor-homing peptide (mUNO, sequence: "CSPGAK") that specifically interacts with mouse CD206 to target CD206/MRC1-expressing tumor-associated macrophages in mice. Here, we report studies on the binding of mUNO to human recombinant CD206 (hCD206) and on modeling the mUNO/hCD206 interaction by computational analysis. Fluorescence anisotropy analysis demonstrated that fluorophore-labeled mUNO interacts with hCD206. Microsecond time-scale molecular dynamics simulations and docking predictions showed that mUNO binds to a newly identified epitope between C-type lectin domains 1 and 2. The physical mechanisms that contribute to the docking interactions of mUNO include electrostatic interactions, aromatic interactions, and hydrogen bonds. We also demonstrate the selectivity of FAM-mUNO for CD206+-cultured human macrophages. The peptide mUNO appears to be the first ligand capable of interacting with this epitope of hCD206, for which no ligands have been reported. Our study has implications for targeting human M2-like tumor-associated macrophages, a subpopulation of immune cells with a major protumoral role.

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.

Competition of charge-mediated and specific binding by peptide-tagged cationic liposome-DNA nanoparticles in vitro and in vivo.

Cationic liposome-nucleic acid (CL-NA) complexes, which form spontaneously, are a highly modular gene delivery system. These complexes can be sterically stabilized via PEGylation [PEG: poly (ethylene glycol)] into nanoparticles (NPs) and targeted to specific tissues and cell types via the conjugation of an affinity ligand. However, there are currently no guidelines on how to effectively navigate the large space of compositional parameters that modulate the specific and nonspecific binding interactions of peptide-targeted NPs with cells. Such guidelines are desirable to accelerate the optimization of formulations with novel peptides. Using PEG-lipids functionalized with a library of prototypical tumor-homing peptides, we varied the peptide density and other parameters (binding motif, peptide charge, CL/DNA charge ratio) to study their effect on the binding and uptake of the corresponding NPs. We used flow cytometry to quantitatively assess binding as well as internalization of NPs by cultured cancer cells. Surprisingly, full peptide coverage resulted in less binding and internalization than intermediate coverage, with the optimum coverage varying between cell lines. In, addition, our data revealed that great care must be taken to prevent nonspecific electrostatic interactions from interfering with the desired specific binding and internalization. Importantly, such considerations must take into account the charge of the peptide ligand as well as the membrane charge density and the CL/DNA charge ratio. To test our guidelines, we evaluated the in vivo tumor selectivity of selected NP formulations in a mouse model of peritoneally disseminated human gastric cancer. Intraperitoneally administered peptide-tagged CL-DNA NPs showed tumor binding, minimal accumulation in healthy control tissues, and preferential penetration of smaller tumor nodules, a highly clinically relevant target known to drive recurrence of the peritoneal cancer.

Application of polymersomes engineered to target p32 protein for detection of small breast tumors in mice.

Triple negative breast cancer (TNBC) is the deadliest form of breast cancer and its successful treatment critically depends on early diagnosis and therapy. The multi-compartment protein p32 is overexpressed and present at cell surfaces in a variety of tumors, including TNBC, specifically in the malignant cells and endothelial cells, and in macrophages localized in hypoxic areas of the tumor. Herein we used polyethylene glycol-polycaprolactone polymersomes that were affinity targeted with the p32-binding tumor penetrating peptide LinTT1 (AKRGARSTA) for imaging of TNBC lesions. A tyrosine residue was added to the peptide to allow for 124I labeling and PET imaging. In a TNBC model in mice, systemic LinTT1-targeted polymersomes accumulated in early tumor lesions more than twice as efficiently as untargeted polymersomes with up to 20% ID/cc at 24 h after administration. The PET-imaging was very sensitive, allowing detection of tumors as small as ∼20 mm3. Confocal imaging of tumor tissue sections revealed a high degree of vascular exit and stromal penetration of LinTT1-targeted polymersomes and co-localization with tumor-associated macrophages. Our studies show that systemic LinTT1-targeted polymersomes can be potentially used for precision-guided tumor imaging and treatment of TNBC.

Targeting of p32 in peritoneal carcinomatosis with intraperitoneal linTT1 peptide-guided pro-apoptotic nanoparticles.

Gastrointestinal and gynecological malignancies disseminate in the peritoneal cavity - a condition known as peritoneal carcinomatosis (PC). Intraperitoneal (IP) administration can be used to improve therapeutic index of anticancer drugs used for PC treatment. Activity of IP anticancer drugs can be further potentiated by encapsulation in nanocarriers and/or affinity targeting with tumor-specific affinity ligands, such as tumor homing peptides. Here we evaluated a novel tumor penetrating peptide, linTT1 (AKRGARSTA), as a PC targeting ligand for nanoparticles. We first demonstrated that the primary homing receptor for linTT1, p32 (or gC1qR), is expressed on the cell surface of peritoneal carcinoma cell lines of gastric (MKN-45P), ovarian (SKOV-3), and colon (CT-26) origin, and that peritoneal tumors in mice and clinical peritoneal carcinoma explants express p32 protein accessible from the IP space. Iron oxide nanoworms (NWs) functionalized with the linTT1 peptide were taken up and routed to mitochondria in cultured PC cells. NWs functionalized with linTT1 peptide in tandem with a pro-apoptotic [D(KLAKLAK)2] peptide showed p32-dependent cytotoxicity in MKN-45P, SKOV-3, and CT-26 cells. Upon IP administration in mice bearing MKN-45P, SKOV-3, and CT-26 tumors, linTT1-functionalized NWs showed robust homing and penetration into malignant lesions, whereas only a background accumulation was seen in control tissues. In tumors, the linTT1-NW accumulation was seen predominantly in CD31-positive blood vessels, in LYVE-1-positive lymphatic structures, and in CD11b-positive tumor macrophages. Experimental therapy of mice bearing peritoneal MKN-45P xenografts and CT-26 syngeneic tumors with IP linTT1-D(KLAKLAK)2-NWs resulted in significant reduction of weight of peritoneal tumors and significant decrease in the number of metastatic tumor nodules, whereas treatment with untargeted D(KLAKLAK)2-NWs had no effect. Our data show that targeting of p32 with linTT1 tumor-penetrating peptide improves tumor selectivity and antitumor efficacy of IP pro-apoptotic NWs. P32-directed intraperitoneal targeting of other anticancer agents and nanoparticles using peptides and other affinity ligands may represent a general strategy to increase their therapeutic index.