Untargeted Liposomes Research Articles

Abstract 4160: V7 targeted liposomes detect pancreatic adenocarcinoma via multispectral optoacoustic tomography

Abstract Purpose: Pancreatic ductal adenocarcinoma (PDA) remains one of the most aggressive malignancies with a 5-year survival rate of 9-10%. Insufficient early detection coupled with limitations in operating surgery renders PDA as one of the most challenging tumors to treat. Accurate localization of tumor margins can tremendously improve surgical treatment outcomes in patients. Recently, nanotechnology has emerged as a promising platform for theranostic applications in the field of drug discovery, although the clinical success is limited by their tumor-specific uptake. Hypoxia due to aberrant vascularization is a hallmark of PDA and can be exploited for active targeting. pH-low insertion peptide variant 7 (V7) is used as hypoxia and acidosis-dependent biomarker for selective targeting of pancreatic cancer tissue. This work is focused on using IR 780 contrast agent loaded, V7 peptide conjugated liposomes for the active targeting of PDA towards enhanced surgical outcomes. Methods: Liposomes encapsulating IR780 dye were prepared using thin film hydration technique. V7 peptide was conjugated to liposomes using an SMCC linker and liposomes were characterized for their size, polydispersity and surface potential. In-vitro uptake of IR780-V7-liposome by S2VP10 at pH 7.4, 6.8, and 6.6 was evaluated through NIR fluorescence imaging using an Odyssey Infrared System. Athymic mice were orthotopically implanted with S2VP10 cells. After tumors were 3 mm, V7-liposomes and untargeted liposomes (0.01 OD) were intravenously injected and biodistribution and tumor specific uptake was accessed using multispectral optoacoustic imaging (MSOT). Results: Dynamic light scattering (DLS) revealed the size of liposomes to be 109 nm with a polydispersity index of 0.198 and zeta potential of +35.8 mV. Surface conjugation of V7 peptide was confirmed using the zeta potential measurement. NIR fluorescence imaging in S2VP10 cells established enhanced tumor specific uptake of V7 conjugated liposomes than passively targeted liposomes (p<0.05). Biodistribution of V7-liposomes demonstrated tumor specific accumulation (PDAC tumor 23.7 a.u, liver 3.6 a.u., and kidney 1.1 a.u.) while untargeted liposomes had the greatest uptake in liver (PDAC tumor 2.1 a.u., liver 6.3 a.u., and kidney 0.7 a.u.) p<0.05. Conclusion: pH-specific actively targeted liposomes can be used to specifically target PDA with minimal off-target binding effects. V7-IR780-liposome formulation can be used to efficiently deliver contrast agent for imaging tumor margins and to expand the potential of MSOT towards clinical applications. Citation Format: Happy Agarwal, Phillip Chuong, William E. Grizzle, Barish H. Edil, Ajay Jain, Lacey R. McNally. V7 targeted liposomes detect pancreatic adenocarcinoma via multispectral optoacoustic tomography [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4160.

Development of PET Radioisotope Copper-64-Labeled Theranostic Immunoliposomes for EGFR Overexpressing Cancer-Targeted Therapy and Imaging.

Combining standard surgical procedures with personalized chemotherapy and the continuous monitoring of cancer progression is necessary for effective NSCLC treatment. In this study, we developed liposomal nanoparticles as theranostic agents capable of simultaneous therapy for and imaging of target cancer cells. Copper-64 (64Cu), with a clinically practical half-life (t1/2 = 12.7 h) and decay properties, was selected as the radioisotope for molecular PET imaging. An anti-epidermal growth factor receptor (anti-EGFR) antibody was used to achieve target-specific delivery. Simultaneously, the chemotherapeutic agent doxorubicin (Dox) was encapsulated within the liposomes using a pH-gradient method. The conjugates of 64Cu-labeled and anti-EGFR antibody-conjugated micelles were inserted into the doxorubicin-encapsulating liposomes via a post-insertion procedure (64Cu-Dox-immunoliposomes). We evaluated the size and zeta-potential of the liposomes and analyzed target-specific cell binding and cytotoxicity in EGFR-positive cell lines. Then, we analyzed the specific therapeutic effect and PET imaging of the 64Cu-Dox-immunoliposomes with the A549 xenograft mouse model. In vivo therapeutic experiments on the mouse models demonstrated that the doxorubicin-containing 64Cu-immunoliposomes effectively inhibited tumor growth. Moreover, the 64Cu-immunoliposomes provided superior in vivo PET images of the tumors compared to the untargeted liposomes. We suggest that nanoparticles will be the potential platform for cancer treatment as a widely applicable theranostic system.

Delivery of RGD-modified liposome as a targeted colorectal carcinoma therapy and its autophagy mechanism

Liposomes are among the most extensively applied drug carriers due to their excellent biocompatibility, controllable size and ease of modification. In the present study, we prepared untargeted liposomes (LP) and targeting liposomes modified with Arg-Gly-Asp (RGD-LP), and Doxorubicin Hydrochloride (DOX) or fluorescent probe was loaded. RGD-LP/DOX was identified to be uniformly spherical in size 131.2 ± 2.7 nm. Based on flow cytometry analysis and the confocal laser scanning microscopy, RGD-LP had a higher uptake into HRT-18 colorectal cancer cells than LP. Further, in vivo imaging study further suggested that RGD-LP could significantly increase the liposome accumulation in the tumour tissues of the mice bearing subcutaneous tumours. By investigating the targeting mechanism of RGD-LP, we found that they entered the cell via macropinocytosis. When loaded with DOX, RGD-LP exerted stronger tumour growth inhibitory activity against tumours of colorectal carcinoma compared to LP. Moreover, RGD-LP induced autophagy. Therefore, RGD-LP have the potential to be applied as a targeted colorectal carcinoma therapy.

Abstract A101: Nucleolin: A novel cell surface protein for neuroblastoma targeted therapy

Abstract Background: Neuroblastoma (NB) represents the most frequent and aggressive form of extracranial solid tumor of infants. Since the use of targeted therapies is severely limited by the lack of specific receptors, chemotherapy and radiotherapy remain the main treatment choices, which, nevertheless expose high-risk NB patients to unacceptable toxicity and to the development of drug resistance. Nucleolin (NCL), a multifunctional protein involved in both physiological and pathological conditions, is overexpressed and partially localized on the cell surface of tumor cells of adult cancers. Little is reported about NCL in pediatric tumors, including NB. Methods: To test the expression of NCL as a potential and targetable cell surface marker in NB, human NB cell lines were evaluated in vitro and after injection in mice, by Imaging Flow Cytometry (IFC) and immunohistochemistry (IHC) analyses, respectively. Cell binding and internalization of rhodamine-labeled nanocarriers, decorated with the NCL-recognizing F3 peptide (Rhod-F3-SL) were evaluated in vitro by FACS and confocal microscopy. The cytotoxic potential of a doxorubicin (DXR)-loaded, pH-sensitive nanocarrier and decorated with the F3 peptide was evaluated in vitro, in terms of inhibition of NB cells proliferation and induction of cell death by CSFE and MTS assays, respectively. Orthotopic and pseudometastatic animal models of human NB, mimicking the growth and spread of NB in patients, were finally enrolled to examine in vivo the anti-tumor activity of DXR-loaded, pH-sensitive nanocarriers decorated with F3 peptide. Results: IFC analyses demonstrated that NCL is expressed at different extent on the surface of all NB cells analyzed. Notably, IHC evaluations displayed NCL staining on both tumor and endothelial tumor cells in well-established NB xenografts. Moreover, in vitro binding studies showed that Rhod-F3-SL strongly associated to NB cells, exhibiting a specific co-localization with cell surface NCL. The cellular distribution of Rhod-F3-SL also confirms the binding specificity, showing an increased selective internalization of NCL-targeted nanoparticles, compared to that obtained with untargeted formulations. Finally, F3-targeted, DXR-loaded, pH-sensitive nanocarriers resulted significantly more effective, in terms of inhibition of cell proliferation and reduction of cell viability in vitro, and in terms of delay of tumor growth in each NB animal model tested, when compared to DXR-loaded, pH-sensitive untargeted liposomes. Conclusion: Our findings demonstrate that NCL represents a novel cell surface protein to be targeted by “drug”-loaded nanoparticles as an innovative therapeutic strategy for high-risk NB. Citation Format: Brignole Chiara, Veronica Bensa, Genny Del Zotto, Silvia Bruno, Nuno A Fonseca, Ana F Cruz, Daniela Di Paolo, Patrizia Perri, Vera Moura, Laura Emionite, Michele Cilli, Mirco Ponzoni, Joao N Moreira, Fabio Pastorino. Nucleolin: A novel cell surface protein for neuroblastoma targeted therapy [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A101. doi:10.1158/1535-7163.TARG-19-A101

Dectin-2-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy.

Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus cause life-threatening candidiasis, cryptococcosis, and aspergillosis, resulting in several hundred thousand deaths annually. The patients at the greatest risk of developing these life-threatening invasive fungal infections have weakened immune systems. The vulnerable population is increasing due to rising numbers of immunocompromised individuals as a result of HIV infection or immunosuppressed individuals receiving anticancer therapies and/or stem cell or organ transplants. While patients are treated with antifungals such as amphotericin B, all antifungals have serious limitations due to lack of sufficient fungicidal effect and/or host toxicity. Even with treatment, 1-year survival rates are low. We explored methods of increasing drug effectiveness by designing fungicide-loaded liposomes specifically targeted to fungal cells. Most pathogenic fungi are encased in cell walls and exopolysaccharide matrices rich in mannans. Dectin-2 is a mammalian innate immune membrane receptor that binds as a dimer to mannans and signals fungal infection. We coated amphotericin-loaded liposomes with monomers of Dectin-2's mannan-binding domain, sDectin-2. sDectin monomers were free to float in the lipid membrane and form dimers that bind mannan substrates. sDectin-2-coated liposomes bound orders of magnitude more efficiently to the extracellular matrices of several developmental stages of C. albicans, C. neoformans, and A. fumigatus than untargeted control liposomes. Dectin-2-coated amphotericin B-loaded liposomes reduced the growth and viability of all three species more than an order of magnitude more efficiently than untargeted control liposomes and dramatically decreased the effective dose. Future efforts focus on examining pan-antifungal targeted liposomal drugs in animal models of fungal diseases.IMPORTANCE Invasive fungal diseases caused by Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus have mortality rates ranging from 10 to 95%. Individual patient costs may exceed $100,000 in the United States. All antifungals in current use have serious limitations due to host toxicity and/or insufficient fungal cell killing that results in recurrent infections. Few new antifungal drugs have been introduced in the last 2 decades. Hence, there is a critical need for improved antifungal therapeutics. By targeting antifungal-loaded liposomes to α-mannans in the extracellular matrices secreted by these fungi, we dramatically reduced the effective dose of drug. Dectin-2-coated liposomes loaded with amphotericin B bound 50- to 150-fold more strongly to C. albicans, C. neoformans, and A. fumigatus than untargeted liposomes and killed these fungi more than an order of magnitude more efficiently. Targeting drug-loaded liposomes specifically to fungal cells has the potential to greatly enhance the efficacy of most antifungal drugs.

A Peptide Probe Enables Photoacoustic-Guided Imaging and Drug Delivery to Lung Tumors in K-rasLA2 Mutant Mice.

The lack of molecular targets and targeting probes remains a major drawback for targeted imaging and drug delivery in lung cancer. In this study, we exploited in vivo phage display to identify a novel targeting probe that homes to the tumor in a K-rasLA2 mutant mouse lung cancer model. Compared with other candidate peptides selected from 5 rounds of phage display, the CRQTKN peptide homed to tumor nodules in the lung of mutant mice at higher levels. Photoacoustic tomography of mutant mice detected lung tumors via tumor homing of the near-infrared fluorescence dye-labeled CRQTKN peptide. Ex vivo photoacoustic images of isolated organs further demonstrated tumor homing of the CRQTKN peptide, whereas minimal accumulation was observed in control organs, such as the liver. Compared with untargeted liposomes and doxorubicin, doxorubicin-loaded liposomes whose surface was modified with the CRQTKN peptide more efficiently delivered doxorubicin and reduced the number or size of tumor lesions in K-rasLA2 mutant mice. Analysis of hematologic parameters and liver and kidney function showed no significant systemic side effects by the treatments. Affinity-based identification was used to detect TNF receptor superfamily member 19L (TNFRSF19L), which was upregulated in lung tumors of mutant mice, as the receptor for the CRQTKN peptide. In conclusion, these results suggest that the CRQTKN peptide is a promising targeting probe for photoacoustic-guided detection and drug delivery to lung cancer, and acts by binding to TNFRSF19L. SIGNIFICANCE: These findings present a new tumor-targeting probe for photoacoustic-guided detection and drug delivery.

Dectin-1-Targeted Antifungal Liposomes Exhibit Enhanced Efficacy.

Aspergillus species cause pulmonary invasive aspergillosis resulting in nearly 100,000 deaths each year. Patients at the greatest risk of developing life-threatening aspergillosis have weakened immune systems and/or various lung disorders. Patients are treated with antifungals such as amphotericin B (AmB), caspofungin acetate, or triazoles (itraconazole, voriconazole, etc.), but these antifungal agents have serious limitations due to lack of sufficient fungicidal effect and human toxicity. Liposomes with AmB intercalated into the lipid membrane (AmB-LLs; available commercially as AmBisome) have severalfold-reduced toxicity compared to that of detergent-solubilized drug. However, even with the current antifungal therapies, 1-year survival among patients is only 25 to 60%. Hence, there is a critical need for improved antifungal therapeutics. Dectin-1 is a mammalian innate immune receptor in the membrane of some leukocytes that binds as a dimer to beta-glucans found in fungal cell walls, signaling fungal infection. Using a novel protocol, we coated AmB-LLs with Dectin-1's beta-glucan binding domain to make DEC-AmB-LLs. DEC-AmB-LLs bound rapidly, efficiently, and with great strength to Aspergillus fumigatus and to Candida albicans and Cryptococcus neoformans, highly divergent fungal pathogens of global importance. In contrast, untargeted AmB-LLs and bovine serum albumin (BSA)-coated BSA-AmB-LLs showed 200-fold-lower affinity for fungal cells. DEC-AmB-LLs reduced the growth and viability of A. fumigatus an order of magnitude more efficiently than untargeted control liposomes delivering the same concentrations of AmB, in essence decreasing the effective dose of AmB. Future efforts will focus on examining pan-antifungal targeted liposomal drugs in animal models of disease.IMPORTANCE The fungus Aspergillus fumigatus causes pulmonary invasive aspergillosis resulting in nearly 100,000 deaths each year. Patients are often treated with antifungal drugs such as amphotericin B (AmB) loaded into liposomes (AmB-LLs), but all antifungal drugs, including AmB-LLs, have serious limitations due to human toxicity and insufficient fungal cell killing. Even with the best current therapies, 1-year survival among patients with invasive aspergillosis is only 25 to 60%. Hence, there is a critical need for improved antifungal therapeutics. Dectin-1 is a mammalian protein that binds to beta-glucan polysaccharides found in nearly all fungal cell walls. We coated AmB-LLs with Dectin-1 to make DEC-AmB-LLs. DEC-AmB-LLs bound strongly to fungal cells, while AmB-LLs had little affinity. DEC-AmB-LLs killed or inhibited A. fumigatus 10 times more efficiently than untargeted liposomes, decreasing the effective dose of AmB. Dectin-1-coated drug-loaded liposomes targeting fungal pathogens have the potential to greatly enhance antifungal therapeutics.

Interleukin-4 receptor as a cancer-related prognosis biomarker and therapeutic target

There are two types of interleukin-4 receptor (IL-4R). Type I IL-4R, a heterodimer of IL-4Rα and IL-2RγC, is expressed on cells of hematopoietic origin such as T cells and B cells. In contrast, type II IL-4R, a heterodimer of IL4Rα and IL-13Ra1 is commonly up-regulated in many types of cancer cells including breast cancer and head and neck cancer. Interaction of IL-4 and IL-4R up-regulates anti-apoptotic proteins such as Bcl-xL in tumor cells. In this study, we examined the co-relationship between IL-4R expression and prognosis of patients. In breast cancer patients, high expression levels of IL-13Ra1 subunit of IL-4R was correlated with low overall survival (OS) and disease-free survival (DFS) compared to negative or low expression of IL-13Ra1. However, in oral squamous cell carcinoma patients, high IL-13Ra1 expression was correlated with increased DFS and cancer-specific survival, while high expression of 13Ra1 was correlated with a lower DFS. Next, we tested the feasibility of IL-4R as a therapeutic target IL-4RPep-1 (IL-4 receptor targeting peptide-1), CRKRLDRNC, bound to H226 lung tumor cells that over-express IL4R, while little binding was observed in H460 lung tumor cells that express IL-4R at low levels. When injected intravenously into nude mice bearing a subcutaneous H226 tumor, Cy7.5 near-infrared fluorescence dye-conjugated IL-4RPep-1 selectively homed to tumor. IL-4RPep-1-labeled liposomes containing doxorubicin showed more efficient anti-tumor growth activity than untargeted liposomes. These findings suggest that IL-4R is a promising cancer-related prognosticable biomarker and also therapeutic target for the treatment of cancer.

A Novel DNA Aptamer for Dual Targeting of Polymorphonuclear Myeloid-derived Suppressor Cells and Tumor Cells.

Aptamers have the potential to be used as targeting ligands for cancer treatment as they form unique spatial structures.Methods: In this study, a DNA aptamer (T1) that accumulates in the tumor microenvironment was identified through in vivo selection and validation in breast cancer models. The use of T1 as a targeting ligand was evaluated by conjugating the aptamer to liposomal doxorubicin.Results: T1 exhibited a high affinity for both tumor cells and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). Treatment with T1 targeted doxorubicin liposomes triggered apoptosis of breast cancer cells and PMN-MDSCs. Suppression of PMN-MDSCs, which serve an immunosuppressive function, leads to increased intratumoral infiltration of cytotoxic T cells. Conclusion: The cytotoxic and immunomodulatory effects of T1-liposomes resulted in superior therapeutic efficacy compared to treatment with untargeted liposomes, highlighting the promise of T1 as a targeting ligand in cancer therapy.

Nano-technology based carriers for nitrogen-containing bisphosphonates delivery as sensitisers of γδ T cells for anticancer immunotherapy.

Nitrogen containing bisphosphonates (N-BPs) including zoledronate (ZOL) and alendronate (ALD) inhibit farnesyl diphosphate synthase, and have been shown to have a cytotoxic affect against cancer cells as a monotherapy and to also sensitise tumour cells to destruction by γδ T cells. γδ T cells are a subset of human T lymphocytes and have a diverse range of roles in the immune system including the recognition and destruction of cancer cells. This property of γδ T cells can be harnessed for use in cancer immunotherapy through in vivo expansion or the adoptive transfer of ex vivo activated γδ T cells. The use of N-BPs with γδ T cells has been shown to have a synergistic effect in in vitro, animal and clinical studies. N-BPs have limited in vivo activity due to rapid clearance from the circulation. By encapsulating N-BPs in liposomes (L) it is possible to increase the levels of N-BPs at non-osseous tumour sites. L-ZOL and L-ALD have been shown to have different toxicological profiles than free ZOL or ALD. Both L-ALD and L-ZOL led to increased spleen weight, leucocytosis, neutrophilia and lymphocytopenia in mice after intravenous injection. L-ALD was shown to be better tolerated than L-ZOL in murine studies. Biodistribution studies have been performed in order to better understand the interaction of N-BPs and γδ T cells in vivo. Additionally, in vivo therapy studies have shown that mice treated with both L-ALD and γδ T cells had a significant reduction in tumour growth compared to mice treated with L-ALD or γδ T cells alone. The use of ligand-targeted liposomes may further increase the efficacy of this combinatory immunotherapy. Liposomes targeting the αvβ6 integrin receptor using the peptide A20FMDV2 had a greater ability than untargeted liposomes in sensitising cancer cells to destruction by γδ T cells in αvβ6 positive cancer cell lines.

Abstract A47: IL-4 receptor-targeted delivery of liposomal doxorubicin and siRNA to tumor

Abstract Targeted delivery of imaging agents and therapeutics to tumors would provide early detection and increased therapeutic efficacy against cancer. Using phage displayed-random peptide libraries, we have identified IL4RPep-1 (IL-4 receptor-binding peptide-1), CRKRLDRNC, that binds to IL-4 receptor (IL4R). IL4R is over-expressed on many types of cancer cells including lung cancer and breast cancer. Peptides have smaller size and in turn may exert better tissue penetration than bulky antibodies. IL4RPep-1 bound to H226 lung tumor cells that over-express IL4R, while little binding was observed in H460 lung tumor cells that express IL4R at low levels. When injected intravenously into nude mice bearing a subcutaneous H226 tumor, Cy7.5 near-infrared fluorescence dye-conjugated IL4RPep-1 selectively homed to tumor. IL4RPep-1-labeled liposomes containing doxorubicin showed more efficient anti-tumor growth activity than untargeted liposomes. In addition, IL4RPep-1-labeled, branched-type polyethyleneimine nanoparticles containing Bcl-xL siRNA more efficiently knocked down Bcl-xL expression and showed higher cytotoxic effect on cancer cells over-expressing IL4R than cancer cells expressing IL4R at low levels. These results suggest that IL4RPep-1 could be a useful probe for affinity-based imaging and drug delivery to tumor that over-expressing IL4R. Citation Format: Guruprasath Padmanaban, Sri Murugan Poongkavithai Vadevoo, Lianhua Chi, Byung-Heon Lee. IL-4 receptor-targeted delivery of liposomal doxorubicin and siRNA to tumor. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A47. doi:10.1158/1538-7445.CHTME14-A47

Abstract 4475: IL-4 receptor-targeted delivery of liposomal doxorubicin and siRNA to tumor

Abstract Targeted delivery of imaging agents and therapeutics to tumors would provide early detection and increased therapeutic efficacy against cancer. Using phage displayed-random peptide libraries, we have identified IL4RPep-1 (IL-4 receptor-binding peptide-1), CRKRLDRNC, that binds to IL-4 receptor (IL4R). IL4R is over-expressed on many types of cancer cells including lung cancer and breast cancer. Peptides have smaller size and in turn may exert better tissue penetration than bulky antibodies. IL4RPep-1 bound to H226 lung tumor cells that over-express IL4R, while little binding was observed in H460 lung tumor cells that express IL4R at low levels. When injected intravenously into nude mice bearing a subcutaneous H226 tumor, Cy7.5 near-infrared fluorescence (NIRF) dye-conjugated IL4RPep-1 selectively homed to tumor. IL4RPep-1-labeled liposomes containing doxorubicin showed more efficient anti-tumor growth activity than untargeted liposomes. In addition, IL4RPep-1-labeled, branched-type polyethyleneimine nanoparticles containing Bcl-xL siRNA more efficiently knocked down Bcl-xL expression and showed higher cytotoxic effect on cancer cells over-expressing IL4R than cancer cells expressing IL4R at low levels. These results suggest that IL4RPep-1 could be a useful probe for affinity-based imaging and drug delivery to tumor that over-expressing IL4R. Citation Format: Poongkavithai Vadevoo Sri Murugan, Gunassekaran Gowri Rangaswamy, Lianhua Chi, Guruprasath Padmanaban, Moon-Chang Baek, In-San Kim, Rang-Woon Park, Byung-Heon Lee. IL-4 receptor-targeted delivery of liposomal doxorubicin and siRNA to tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4475. doi:10.1158/1538-7445.AM2014-4475

Development of synthetic peptide-modified liposomes with LDL receptor targeting capacity and improved anticancer activity.

In this study, we report an active targeting liposomal formulation directed by a novel peptide (AA13) that specifically binds to the low density lipoprotein receptor (LDLR) overexpressed on acute myeloid leukemia (AML) cells. The objectives of this study were to evaluate the in vitro and in vivo tumor drug targeting delivery of AA13-anchored liposomes on AML cells. AA13 conjugated to the distal end of DSPE-PEG2000-maleimide was incorporated into the liposomes via a postinsertion method. To study the effect of the peptide decoration and density on tumor cell targeting and internalization by AML cells (THP-1 and NB4), stealth liposomes bearing 3% (peptide/S100PC, molar ratio, LL) and 7% (peptide/S100PC, molar ratio, HL) AA13 were prepared, respectively. Higher uptake of LL (1.9- and 2.6-fold) and HL (2.3- and 3.6-fold) targeted liposomes occurred in THP-1 and NB4 cells, respectively, compared to untargeted liposomes. An LDLR inhibitor was used to confirm inhibition of the receptor-mediated cellular association of AA13 modified liposome in both cells. Daunorubicin (DNR) demonstrated a 2.2- and 3.5-fold higher cytotoxicity with the HL formulation and a 1.2- and 2.0-fold higher cytotoxicity with the LL formulation compared to the unmodified liposomal formulation in THP-1 and NB4 cells, respectively. Tumor drug accumulation of DNR-loaded HL was greater than that of the untargeted liposome in the biodistribution assay. The in vivo efficacy study in BALB/c nude mice bearing NB4 xenografts treated with DNR loaded HL also showed more tumor volume inhibition and a longer survival time compared to the untargeted formulation. In conclusion, the AA13-anchored liposomes demonstrated desirable potential as a promising vector for enhanced AML tumor drug targeting.

Enhancement of the uptake and cytotoxic activity of doxorubicin in cancer cells by novel cRGD-semipeptide-anchoring liposomes.

Novel liposemipeptides hanging cyclic azabicycloalkane-RGD or aminoproline-RGD terminals were synthesized and incorporated into liposomal nanoparticles cAba/cAmpRGD-LNP5 3C/3D. Liposomes with similar composition and lacking semipeptide conjugates were constructed for comparison (LNP, 3A), and physical encapsulation of the anticancer doxorubicin drug in both targeted and untargeted liposomes was accomplished. Microstructural analysis performed by dynamic light scattering (DLS), small-angle neutron scattering (SANS), and electron paramagnetic resonance (EPR) revealed that the conjugated nanoparticles presented an average size of 80 nm and were constituted by 5 nm thick unilamellar liposome bilayer. Flow cytometry and fluorescent microscopy studies showed that 3C-DOXO and 3D-DOXO efficiently delivered the drug into the nuclei of both quiescent and proliferating cells even in a high serum concentration environment. The uptake of doxorubicin when carried by liposomes was faster than that of the free drug, and 30 min incubation was sufficient to load cell nuclei with doxorubicin. Targeted liposomes significantly induced cell death of human breast adenocarcinoma MCF7 cells (IC50 = 144 nM, 3C-DOXO; IC50 = 274 nM, 3D-DOXO), about 2- to 6-fold more potent than free doxorubicin or 3A-DOXO controls (IC50 = 527 and 854 nM, respectively). These results suggest that cAba/cAmpRGD liposomal nanoparticles hold promise for the rapid and efficient delivery of chemotherapeutic agents to αVβ3-expressing tumor cells.

Targeting of BRAF resistant melanoma via extracellular matrix metalloproteinase inducer receptor

BackgroundThe BRAF inhibitor vemurafenib (PLX) has shown promise in treating metastatic melanoma, but most patients develop resistance to treatment after 6 mo. We identified a transmembrane protein, extracellular matrix metalloproteinase inducer (EMMPRIN) as a cell surface receptor highly expressed by PLX-resistant melanoma. Using an S100A9 ligand, we created an EMMPRIN targeted probe and liposome that binds to melanoma cells in vivo, thus designing a novel drug delivery vehicle. MethodsPLX-resistant cells were established through continuous treatment with PLX-4032 over the course of 1 y. Both PLX-resistant and sensitive melanoma cell lines were evaluated for the expression of unique cell surface proteins, which identified EMMPRIN as an overexpressed protein in PLX0-resistant cells and S100A9 is a ligand for EMMPRIN. To design a probe for EMMPRIN, S100A9 ligand was conjugated to a CF-750 near-infrared (NIR) dye. EMMPRIN targeted liposomes were created to encapsulate CF-750 NIR dye. Liposomes were characterized by scanning electron microscopy, flow cytometry, and in vivo analysis. A2058PLX and A2058 cells were subcutaneously injected into athymic mice. S100A9 liposomes were intravenously injected and tumor accumulation was evaluated using NIR fluorescent imaging. ResultsWestern blot and flow cytometry demonstrated that PLX sensitive and resistant A2058 and A375 melanoma cells highly express EMMPRIN. S100A9 liposomes were 200 nm diameter and uniformly sized. Flow cytometry demonstrated 100X more intracellular dye uptake by A2058 cells treated with S100A9 liposomes compared with untargeted liposomes. In vivo accumulation of S100A9 liposomes within subcutaneous A2058 and A2058PLX tumors was observed from 6–48 h, with A2058PLX accumulating significantly higher levels (P = 0.001626). ConclusionsEMMPRIN-targeted liposomes via an S100A9 ligand are a novel, targeted delivery system which could provide improved EMMPRIN specific drug delivery to a tumor.

A Liposomal Drug Platform Overrides Peptide Ligand Targeting to a Cancer Biomarker, Irrespective of Ligand Affinity or Density

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for αvβ6-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to αvβ6. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.

Antitumor Activity of Tumor-Targeted RNA Replicase-Based Plasmid That Expresses Interleukin-2 in a Murine Melanoma Model

Double-stranded RNA (dsRNA) has multiple antitumor mechanisms that may be used to control tumor growth. Previously we have shown that treatment of solid tumors with a plasmid that encodes Sindbis viral RNA replicase complex, pSIN-β, significantly inhibited the growth of tumors in mice. In the present study, we evaluated the feasibility of further improving the antitumor activity of the pSIN-β plasmid by incorporating interleukin-2 (IL2) gene into the plasmid. The resultant pSIN-IL2 plasmid was delivered to mouse melanoma cells that overexpress the sigma receptor. Here we report that the pSIN-IL2 plasmid was more effective at controlling the growth of B16 melanoma in mice when complexed with sigma receptor-targeted liposomes than with the untargeted liposomes. Importantly, the pSIN-IL2 plasmid was more effective than pSIN-β plasmid at controlling the growth of B16 melanoma in mice, and B16 tumor-bearing mice that were treated with pSIN-IL2 had an elevated number of activated CD4(+), CD8(+), and natural killer cells, as compared to those treated with pSIN-β. The RNA replicase-based, IL2-expressing plasmid may have applications in melanoma gene therapy.

Folate-targeted PEGylated liposomes improve the selectivity of PDT with meta-tetra(hydroxyphenyl)-chlorin (m-THPC)

The folate receptor (FR) is over-expressed in many human tumours and is being intensively studied also in the field of nanomedicine as a target to enhance the selectivity of drug delivery to cancer cells by using nanocarriers bearing folic acid (FA) on their surface. In this study we report the encapsulation of the photosensitizer (PS) meta-tetra(hydroxyphenyl)chlorin (m-THPC) in FA-targeted PEGylated liposomes used as a novel drug delivery system for photodynamic therapy (PDT) of cancer. Our in vitro investigations revealed that only a modest fraction of targeted liposomes were internalized by specific endocytosis in FR-positive KB cells. However, FA-liposomes doubled the uptake of the entrapped m-THPC with respect to un-targeted liposomes and enhanced the photo-induced cytotoxicity in KB cells. In contrast, in FR-negative A549 cells FA-targeted or un-targeted liposomes exhibited a very similar extent of internalization and as a consequence the same photo-killing efficiency.

Abstract 5485: Apoptosis-targeted cell and drug delivery to tumor.

Abstract When tumor cells undergo apoptosis in response to chemotherapy, the levels of apoptotic biomarkers such as phosphatidylserine and histone H1 are increased at the cell surface. Here we hypothesized that the chemotherapy-induced apoptosis would amplify in situ apoptotic biomarkers (homing signals) for apoptosis-targeted drug carriers and enhance drug delivery. To examine this possibility, we employed a phage display-identified CQRPPR peptide (ApoPep-1) as a targeting moiety, which was able to recognize apoptotic cells by binding to histone H1 on the surface of apoptotic cells. When injected into tumor-bearing mice, ApoPep-1-labeled, fluorescent liposomes containing doxorubicin inhibited tumor growth more efficiently than untargeted or folate-labeled liposomes. Moreover, in vivo fluorescence imaging could enable monitoring of tumor response during the chemotherapy. The imaging signals at tumor were increased by the homing of apoptosis-targeted liposomes, which was correlated with the increase of apoptosis and the amount of doxorubicin (payloads) at the tumor and, conversely, with the decrease of tumor volume. Next, we harnessed the chemotherapy-induced apoptosis of tumor cells as a homing signal for the delivery of apoptosis-targeted T cells to tumor. When labeled with ApoPep-1 using an oleyl acid-derived membrane anchor, targeted T cells preferentially bound to apoptotic tumor cells over living cells. In vivo imaging showed higher levels of tumor homing of targeted, fluorescent T cells in mice treated with chemotherapy more than those of untargeted T cells. These results demonstrate that the apoptosis-targeted delivery can efficiently enhance the delivery of cells or drugs to tumor and, when combined with imaging of apoptosis, provides a real-time monitoring of tumor response for cancer theragnosis. Citation Format: Lianhua Chi, Napoleon Bonaparte, Rang-woon Park, In-san Kim, Byung-heon Lee. Apoptosis-targeted cell and drug delivery to tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5485. doi:10.1158/1538-7445.AM2013-5485

Abstract C90: HER2-targeted liposomal doxorubicin MM-302 has a favorable cardiosafety profile in preclinical models.

Abstract Introduction: Anthracyclines have historically been the backbone of anticancer therapy for decades; however, the clinical observation of permanent cardiotoxicity has limited their broader use. In the HER2 positive breast cancer setting, the combination of trastuzumab with anthracyclines was associated with an unacceptable risk of clinical cardiotoxicity. This appears to be related to 1) doxorubicin-induced cardiomyocyte stress and 2) inability of the cardiomyocytes to respond to the stress due to the trastuzumab-related impairment of HER2 signaling. Encapsulation of doxorubicin into liposomes (Doxil®) has been associated with a reduced risk of doxorubicin-associated cardiotoxicity relative to free doxorubicin. MM-302 is a HER2-targeted liposomal doxorubicin-based agent designed to target doxorubicin to HER2-overexpressing cancer cells, while limiting uptake into non-target cells. Since HER2-mediated signaling plays an important role in cardiac repair, the purpose of this work was to support clinical development of MM-302 by investigating whether HER2-targeting of liposomal doxorubicin would adversely affect the cardiac safety profile relative to untargeted liposomal doxorubicin. Methods: MM-302, untargeted liposomal doxorubicin and free doxorubicin were compared for their uptake into HER2-overexpressing cancer cells and two models of human stem cell-derived cardiomyocytes. High-content microscopy was used to determine the effects of exposure to specific cellular targets in cardiomyocytes. Mouse biodistribution studies were used to assess the total and nuclear accumulation of doxorubicin in mouse cardiac tissue upon treatment with either MM-302, untargeted liposomes or free doxorubicin. Quantitative immunofluorescence was used to quantify the HER2 expression on human normal and diseased heart specimens. Kinetic computational modeling was applied to interpret study results and to make predictions on human heart exposure to doxorubicin based on the experimental mouse data. Results: Human stem cell-derived cardiomyocytes showed nuclear accumulation of doxorubicin followed by cell death upon free doxorubicin treatment but not upon MM-302 or untargeted liposomes treatment. Similarly, MM-302 resulted in a significantly lower nuclear accumulation of doxorubicin compared to free doxorubicin treatment in mouse cardiac tissue. HER2 expression levels on normal and diseased human heart tissue were shown to be ≤100,000 receptors/cell, in concordance with the HER2 levels on the stem cell-derived cardiomyocytes found in vitro. Computational modeling predicts liposomal encapsulation to significantly protect the human heart from exposure to doxorubicin, consistent with clinical findings. Conclusions: MM-302 is not taken up by human cardiomyocytes via HER2, but selectively increases doxorubicin delivery to human HER2-overexpressing tumor cells. MM-302 can potentially improve the clinical efficacy demonstrated by conventional anthracyclines and maintain the cardiac safety profile of untargeted liposomal doxorubicin in patients with HER2 overexpressing cancers. Clinical evaluation of this patient population is currently in Phase I. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C90.