Scott Poh

Development of tumor-targeted near infrared probes for fluorescence guided surgery.

Complete surgical resection of malignant disease is the only reliable method to cure cancer. Unfortunately, quantitative tumor resection is often limited by a surgeons ability to locate all malignant disease and distinguish it from healthy tissue. Fluorescence-guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant lesions. While nontargeted fluorescent dyes have been shown to passively accumulate in some tumors, the resulting tumor-to-background ratios are often poor, and the boundaries between malignant and healthy tissues can be difficult to define. To circumvent these problems, our laboratory has developed high affinity tumor targeting ligands that bind to receptors that are overexpressed on cancer cells and deliver attached molecules selectively into these cells. In this study, we explore the use of two tumor-specific targeting ligands (i.e., folic acid that targets the folate receptor (FR) and DUPA that targets prostate specific membrane antigen (PSMA)) to deliver near-infrared (NIR) fluorescent dyes specifically to FR and PSMA expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here that all FR- and PSMA-targeted NIR probes examined bind cultured cancer cells in the low nanomolar range. Moreover, upon intravenous injection into tumor-bearing mice with metastatic disease, these same ligand-NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues.

A Folate Receptor-α–Specific Ligand That Targets Cancer Tissue and Not Sites of Inflammation

Folic acid has been frequently exploited to target attached drugs to cells that overexpress a folate receptor (FR). Unfortunately, folic acid and folate-linked drugs bind equally well to both major isoforms of the FR—that is, FR-α, which is primarily expressed on malignant cells, and FR-β, which is upregulated on activated monocytes and macrophages. Because both major isoforms of FR can be expressed simultaneously in the same organism, folic acid cannot enable selective targeting of therapeutic and imaging agents to either tumor masses or sites of inflammation. In an effort to develop a targeting ligand that can selectively deliver attached imaging and therapeutic agents to tumor cells, we constructed a reduced and alkylated form of folic acid, N5, N10-dimethyl tetrahydrofolate (DMTHF) that exhibits selectivity for FR-α. Methods: DMTHF–99mTc was injected into mice bearing FR-α–expressing tumor xenografts and imaged by γ-scintigraphy. The selectivity for FR-α over FR-β in vivo was examined by γ-scintigraphic images of animal models of various inflammatory diseases such as apolipoprotein E–deficient mice with atherosclerosis, DBA/1 LacJ mice with induced arthritis, C57BL/6J mice with muscle injury, and BALB/C mice with both FR-α tumor and ulcerative colitis, by administration of equal doses of DMTHF–99mTc and EC20–99mTc. The uptake of radiochelates in various organs was quantified by biodistribution studies. DMTHF–near-infrared dye conjugate and DMTHF–Oregon green dye conjugates were synthesized and evaluated for FR-α selectivity over FR-β in rat peritoneal macrophages and human peripheral blood monocytes, respectively, by flow cytometry. Fluorescence-guided imaging was also performed using folate and DMTHF dye conjugates. Results: The new targeting ligand was found to bind malignant cells in mice with solid tumor xenografts but not peripheral blood monocytes or inflammatory macrophages in animal models of atherosclerosis, rheumatoid arthritis, muscle injury, or ulcerative colitis. Results from optical and radioimaging studies and biodistribution experiments confirm the differential specificity of this new ligand for malignant masses. Conclusion: The new targeting ligand DMTHF enables selective noninvasive imaging and therapy of tumor tissues in the presence of inflammation.

Comparison of nanoparticle penetration into solid tumors and sites of inflammation: Studies using targeted and nontargeted liposomes

AIM The vast majority of nanomedicine research is focused on the use of nanoparticles for the diagnosis and treatment of cancer. However, the dense extracellular matrix of solid tumors restricts nanoparticle penetration, raising the question of whether the best applications of nanomedicines lie in oncology. MATERIALS & METHODS In this study, the uptake of folate-conjugated liposomes was compared between folate receptor-expressing tumors and folate receptor+ inflammatory lesions within the same mouse. RESULTS We demonstrate here that both folate-targeted and nontargeted liposomes accumulate more readily at sites of inflammation than in solid tumors. CONCLUSION These data suggest that nanosized imaging and therapeutic agents may be better suited for the treatment and diagnosis of inflammatory/autoimmune diseases than cancer.

Controlled release of anti-inflammatory peptides from reducible thermosensitive nanoparticles suppresses cartilage inflammation

Characterized by pain, cartilage degradation, and inflammation, osteoarthritis is often treated with anti-inflammatory therapies that provide short-term relief but can have adverse side effects; intra-articular drug delivery systems with controlled release of anti-inflammatory peptides using degradable poly(N-isopropylacrylamide) (pNIPAM) nanoparticles could prolong relief and minimize these side effects. Nanoparticles provide a biocompatible drug carrier that can protect encapsulated therapeutics from enzymatic degradation and increase payload delivery upon encountering a degradation stimulus. Here we demonstrate passive targeting of inflamed cartilage ex vivo by uptake of PEGylated pNIPAM nanoparticles with degradable disulfide crosslinks (abbreviated as NGPEGSS) into chondrocytes and subsequent intracellular release of an anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK). The KAFAK-loaded NGPEGSS treatment reduced ex vivo inflammation to a greater extent compared to its non-degradable counterparts. This study highlights a nanoparticle system that delivers therapeutics intracellularly with improved efficacy by triggered degradation and suppresses inflammation in multiple cell types within an inflamed joint.

Delivery of anti-inflammatory peptides from hollow PEGylated poly(NIPAM) nanoparticles reduces inflammation in an ex vivo osteoarthritis model

ABSTRACT Targeted delivery of anti‐inflammatory osteoarthritis treatments have the potential to significantly decrease undesirable systemic side effects and reduce required therapeutic dosage. Here we present a targeted, non‐invasive drug delivery system to decrease inflammation in an osteoarthritis model. Hollow thermoresponsive poly(N‐isopropylacrylamide) (pNIPAM) nanoparticles have been synthesized via degradation of a N,N′‐bis(acryloyl)cystamine (BAC) cross‐linked core out of a non‐degradable pNIPAM shell. Sulfated 2‐acrylamido‐2‐methyl‐1‐propanesulfonic acid (AMPSA) was copolymerized in the shell to increase passive loading of an anti‐inflammatory mitogen‐activated protein kinase‐activated protein kinase 2 (MK2)‐inhibiting cell‐penetrating peptide (KAFAK). The drug‐loaded hollow nanoparticles were effective at delivering a therapeutically active dose of KAFAK to bovine cartilage explants, suppressing pro‐inflammatory interleukin‐6 (IL‐6) expression after interleukin‐1 beta (IL‐1&bgr;) stimulation. This thermosensitive hollow nanoparticle system provides an excellent platform for the delivery of peptide therapeutics into highly proteolytic environments such as osteoarthritis.

Folate-conjugated liposomes target and deliver therapeutics to immune cells in a rat model of rheumatoid arthritis.

AIM We endeavored to create a folate-targeted liposome (Fol-liposome) that could selectively target areas of inflammation. MATERIALS & METHODS Fol-liposomes were prepared with encapsulated DiD fluorophore or betamethasone (BM) to image and treat an adjuvant-induced rat model of rheumatoid arthritis. RESULTS Fol-liposomes selectively accumulated in arthritic rat paws to a greater extent than nontargeted liposomes. When these Fol-liposomes were used to encapsulate BM and administered to arthritic rats, animals exhibited less paw swelling, lower arthritis scores, a reduction in bone erosion, less splenomegaly and better maintenance of body weight when compared with nontreated or nontargeted BM-containing liposome groups. CONCLUSION Fol-liposomes can selectively deliver imaging and therapeutic agents to sites of inflammation in a rat model of rheumatoid arthritis.

Selective liposome targeting of folate receptor positive immune cells in inflammatory diseases

Activated macrophages play a key role in the development and maintenance of inflammatory diseases such as atherosclerosis, lupus, psoriasis, rheumatoid arthritis, ulcerative colitis, and many others. These activated macrophages, but not resting or quiescent macrophages highly up-regulate folate receptor beta (FR-β). This differential expression of FR-β provides a mechanism to selectively deliver imaging and therapeutic agents utilizing folate as a targeting molecule. In an effort to determine whether inflammatory diseases can be targeted utilizing a folate-linked nanosize carrier, a PEG-coated liposome was prepared that incorporated a folate conjugated PEG that also could transport imaging or therapeutic cargo. We demonstrate that these folate-liposomes specifically bind to folate receptor positive cells and accumulate at sites of inflammation in mouse models of colitis and atherosclerosis. These two animal models show that folate-targeted liposomes could be successfully utilized to deliver fluorescent molecules and an anti-inflammatory drug (betamethasone) for diagnostic and therapeutic applications.

Folate-targeted dendrimers selectively accumulate at sites of inflammation in mouse models of ulcerative colitis and atherosclerosis

Folate-receptor-positive activated macrophages are critical for the development and maintenance of many chronic inflammatory and autoimmune diseases. Previously, small-molecule folate-targeted conjugates were found to specifically bind to these activated macrophages in vitro and selectively accumulate at sites of inflammation in vivo. While these small-molecule conjugates have shown promise, the use of a folate-targeted, higher cargo capacity nanovehicle may prove superior in delivering imaging or therapeutic agents in vivo. This nanoparticle strategy has been demonstrated in oncology, where targeted dendrimers have shown superior delivery capabilities; however, little research has been pursued in the area of folate-targeted dendrimers for inflammation and autoimmune diseases. Therefore, we endeavored to create a folate-decorated dendrimer to explore its uptake in mouse models of ulcerative colitis and atherosclerosis. We demonstrate that our final poly(ethylene glycol)-coated, acetic-anhydride-capped, folate-targeted poly(amidoamine) dendrimer exhibits no discernible cytotoxicity in vitro, specifically binds to a folate-receptor-expressing macrophage cell line in vitro, and selectively accumulates in areas of inflammation in vivo.

Suppression of Pro-Inflammatory Cytokine Response by Anti-Inflammatory Peptide Release from Reducible Thermosensitive Nanoparticles

Activated macrophages constitute key players in the development of inflammatory disease such as osteoarthritis. Treatment of inflammatory disorders using peptide drugs may be effective by promoting anti-inflammatory effects. However, poor bioavailability and potential peptide antigenicity may result in unwanted immune responses thus limit its commercial applications. Therefore, in order to improve the efficacy of the peptide drug, alternative delivery approaches must be sought. For this purpose, we designed a reducible thermosensitive biocompatible poly(N-isopropylacrylamide) (pNIPAm) polymer as a drug carrier to protect the peptide from enzymatic degradation, thus increasing the half-life and efficacy of the peptide. The studies presented suggest pNIPAm nanoparticles can be an effective alternative tool for treatment of osteoarthritis ex-vivo model.

Abstract 374: Development of tumor-targeted near infrared dyes for fluorescence guided surgery

Surgical resection of malignant disease is currently one of the most common and effective cancer treatments. Unfortunately, quantitative tumor resection is limited by a surgeon9s ability to distinguish diseased tissue from adjacent healthy tissue. Fluorescence guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant tissue. While non-targeted fluorescent dyes injected near the primary tumor mass have been shown to passively accumulate in tumors and lymph nodes, the resulting tumor to background ratios are often poor and boundaries between malignant and normal tissue difficult to define. To circumvent these problems, our lab has developed high affinity tumor-specific ligands that can bind to either the prostate specific membrane antigen (PSMA) or the folate receptor (FR); i.e. cell surface molecules that are over-expressed on a number of cancers. In this study, we link these tumor targeting ligands to near infrared (NIR) dyes and explore their use in fluorescence guided surgery. In vitro, all FR and PSMA targeted NIR dyes were found to have binding affinities in the low nanomolar range. Binding was quantitatively blocked with 100-fold excess of free ligand. Upon injection into mice with metastatic disease, the targeted NIR dyes allowed clear distinction of tumor tissue from adjacent healthy tissue. Surgical resection could be conducted until all fluorescent tissue was removed. All resected fluorescent lesions were later confirmed by histology to be malignant. Of the NIR dyes tested, the 800 nm dyes were found to have the greatest fluorescence intensity through porcine tissue, indicating their superior ability to reveal buried tumor nodules. Collectively, these tumor-specific NIR dyes demonstrate significant potential for use in fluorescent guided surgery by aiding in the complete resection of diseased tissue. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 374. doi:1538-7445.AM2012-374