Lipid Calcium Phosphate Research Articles

Mannan-Decorated Lipid Calcium Phosphate Nanoparticle Vaccine Increased the Antitumor Immune Response by Modulating the Tumor Microenvironment.

With the rapid development of tumor immunotherapy, nanoparticle vaccines have attracted much attention as potential therapeutic strategies. A systematic review and analysis must be carried out to investigate the effect of mannose modification on the immune response to nanoparticles in regulating the tumor microenvironment, as well as to explore its potential clinical application in tumor therapy. Despite the potential advantages of nanoparticle vaccines in immunotherapy, achieving an effective immune response in the tumor microenvironment remains a challenge. Tumor immune escape and the overexpression of immunosuppressive factors limit its clinical application. Therefore, our review explored how to intervene in the immunosuppressive mechanism in the tumor microenvironment through the use of mannan-decorated lipid calcium phosphate nanoparticle vaccines to improve the efficacy of immunotherapy in patients with tumors and to provide new ideas and strategies for the field of tumor therapy.

Dexamethasone-Loaded Lipid Calcium Phosphate Nanoparticles Treat Experimental Colitis by Regulating Macrophage Polarization in Inflammatory Sites.

The M1/M2 polarization of intestinal macrophages exerts an essential function in the pathogenesis of ulcerative colitis (UC), which can be adjusted to alleviate the UC symptoms. A kind of pH-sensitive lipid calcium phosphate core-shell nanoparticles (NPs), co-loading with dexamethasone (Dex) and its water-soluble salts, dexamethasone sodium phosphate (Dsp), was constructed to comprehensively regulate macrophages in different states towards the M2 phenotype to promote anti-inflammatory effects. Dex and Dsp were loaded in the outer lipid shell and inner lipid calcium phosphate (Cap) core of the LdCaPd NPs, respectively. Then, the morphology of NPs and methods for determining drug concentration were investigated, followed by in vitro protein adsorption, stability, and release tests. Cell experiments evaluated the cytotoxicity, cellular uptake, and macrophage polarization induction ability of NPs. The in vivo distribution and anti-inflammatory effect of NPs were evaluated through a 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced BALB/c mice ulcerative colitis model. The LdCaPd NPs showed a particle size of about 200 nm and achieved considerable loading amounts of Dex and Dsp. The in vitro and in vivo studies revealed that in the acidic UC microenvironment, the cationic lipid shell of LdCaPd underwent protonated dissociation to release Dex first for creating a microenvironment conducive to M2 polarization. Then, the exposed CaP core was further engulfed by M1 macrophages to release Dsp to restrict the pro-inflammatory cytokines production by inhibiting the activation and function of the nuclear factor kappa-B (NF-κB) through activating the GC receptor and the NF kappa B inhibitor α (I-κBα), respectively, ultimately reversing the M1 polarization to promote the anti-inflammatory therapy. The LdCaPd NPs accomplished the sequential release of Dex and Dsp to the UC site and the inflammatory M1 macrophages at this site, promoting the regulation of macrophage polarization to accelerate the remission of UC symptoms.

STING agonist delivery by lipid calcium phosphate nanoparticles enhances immune activation for neuroblastoma

Neuroblastoma (NB) is a common solid tumor in children and infants, the formation and regression of which is closely linked to the tumor-host immune relationship. Stimulator of interferon genes (STING) agonists, particularly cyclic dinucleotide (CDN), have promising potential in NB therapy by generating innate and adaptive immune stimulation, thus leading to tumor control. CDN delivery in vivo is challenging due to the negative charge, hydrophilicity, and susceptibility to degradation by phosphodiesterase, which hinders the effectiveness of CDN. Thus, our study proposed four methods to load CDN into liposomes, using 2′,3′-cGAMP as the model drug. Lipid nanoparticles were prepared, followed by physicochemical characterization. Subsequently, cellular inhibition and immune stimulation were investigated. As a result, lipid calcium phosphate nanoparticles (LCP-NPs) possessed the highest encapsulation efficiency among the four preparation methods, with a diameter of 82.57±3.72 nm. LCP-NPs maintained size stability under refrigeration conditions at 4°C within 48 h. The surface of the liposome was positively charged. Compared to free cGAMP, LCP-NPs resulted in a slower release, enhanced cytotoxicity against tumor cells, greater activation of the cGAS-STING pathway, and increased expression of the immune factors. Taken together, these findings clearly demonstrated the effectiveness of the liposomal delivery system for cGAMP and provided a promising strategy for the treatment of NB.

Nanoparticle Delivery of miR-122 Inhibits Colorectal Cancer Liver Metastasis.

Liver metastasis is a leading cause of cancer morbidity and mortality. Thus, there has been strong interest in the development of therapeutics that can effectively prevent liver metastasis. One potential strategy is to utilize molecules that have broad effects on the liver microenvironment, such as miR-122, a liver-specific miRNA that is a key regulator of diverse hepatic functions. Here we report the development of a nanoformulation miR-122 as a therapeutic agent for preventing liver metastasis. We engineered a galactose-targeted lipid calcium phosphate (Gal-LCP) nanoformulation of miR-122. This nanotherapeutic elicited no significant toxicity and delivered miR-122 into hepatocytes with specificity and high efficiency. Across multiple colorectal cancer liver metastasis models, treatment with Gal-LCP miR-122 treatment effectively prevented colorectal cancer liver metastasis and prolonged survival. Mechanistic studies revealed that delivery of miR-122 was associated with downregulation of key genes involved in metastatic and cancer inflammation pathways, including several proinflammatory factors, matrix metalloproteinases, and other extracellular matrix degradation enzymes. Moreover, Gal-LCP miR-122 treatment was associated with an increased CD8+/CD4+ T-cell ratio and decreased immunosuppressive cell infiltration, which makes the liver more conducive to antitumor immune response. Collectively, this work presents a strategy to improve cancer prevention and treatment with nanomedicine-based delivery of miRNA. SIGNIFICANCE: Highly specific and efficient delivery of miRNA to hepatocytes using nanomedicine has therapeutic potential for the prevention and treatment of colorectal cancer liver metastasis.

Novel Pyropheophorbide Phosphatydic Acids Photosensitizer Combined EGFR siRNA Gene Therapy for Head and Neck Cancer Treatment.

This study combined two novel nanomedicines, a novel LCP Pyro PA photodynamic therapy (PDT) and LCP EGFR siRNA gene therapy, to treat head and neck cancer. A novel photosensitizer, pyropheophorbide phosphatydic acids (Pyro PA), was first modified into Lipid-Calcium phosphate nanoparticles named LCP Pyro PA NPs, and targeted with aminoethylanisamide as a novel PDT photosensitizer. EGFR siRNA was encapsulated into LCP NPs to silence EGFR expression. Measured sizes of LCP EGFR siRNA NPs and LCP Pyro-PA NPs were 34.9 ± 3.0 and 20 nm respectively, and their zeta potentials were 51.8 ± 1.8 and 52.0 ± 7.6 mV respectively. In vitro studies showed that EGFR siRNA was effectively knocked down after photodynamic therapy (PDT) with significant inhibition of cancer growth. SCC4 or SAS xenografted nude mice were used to verify therapeutic efficacy. The LCP Control siRNA+PDT group of SCC4 and SAS showed significantly reduced tumor volume compared to the phosphate buffered saline (PBS) group. In the LCP-EGFR siRNA+LCP Pyro PA without light group and LCP EGFR siRNA + PBS with light group, SCC4 and SAS tumor volumes were reduced by ~140% and ~150%, respectively, compared to the PBS group. The LCP EGFR siRNA+PDT group of SCC4 and SAS tumor volumes were reduced by ~205% and ~220%, respectively, compared to the PBS group. Combined therapy showed significant tumor volume reduction compared to PBS, control siRNA, or PDT alone. QPCR results showed EGFR expression was significantly reduced after treatment with EGFR siRNA with PDT in SCC4 and SAS compared to control siRNA or PDT alone. Western blot results confirmed decreased EGFR protein expression in the combined therapy group. No toxic results were found in serum biomarkers. No inflammatory factors were found in heart, liver and kidney tissues. Results suggest that the novel LCP Pyro PA mediated PDT combined with LCP siEGFR NPs could be developed in clinical modalities for treating human head and neck cancer in the future.

Preparation and Characterization of siRNA-Loaded Liposomes.

The major challenge for RNAi-based therapy is the fabrication of the delivery system that meet the requirement of clinical applicability. Liposome-derived nanoparticles (NPs) are one of the best investigated systems for in vivo siRNA delivery. In the recent years, we have successfully redesigned the conventional cationic liposomes into Liposome/Protamine/hyaluronic acid (LPH) NPs and Lipid-Calcium-Phosphate (LCP) NPs in order to increase the in vivo gene silencing effect and reduce the toxicity. Here we describe the preparation of LPH and LCP NPs loaded with siRNA, and characterization analysis including size distribution, trapping efficiency, and in vivo activity. This protocol could be used for in vivo delivery of siRNA to target genes in cancer cells.

Abstract 5033: Targeted delivery of miR-122 into hepatocytes regresses liver metastasis

Abstract Background: Metastatic tumors in the liver are 20 times more common than primary tumors and considered as a leading cause of mortality in several types of cancer. Therefore, making the liver (soil) inhospitable for tumor cells (seeds) to grow will have tremendous effects on outcome of multiple types of cancer. RNAi delivery -including miRNA delivery-shows promise for the development of novel molecular therapeutics that interfere with genes causing metastasis. Mir-122, which accounts for 70% of total miRNA population within the liver, is also known as a tumor suppressor miRNA within liver. We hypothesized that miR-122 delivery into hepatocytes can prevent development of metastatic colorectal cancer within mice liver. For testing this hypothesis, we used hepatic injection of ex-vivo engineered liver metastasis from human colorectal cancer cells to induce hepatic metastasis in nude mice and employed lipid calcium phosphate (LCP) nanoparticles for delivery of miR-122. Methods: LCP-miR122 and LCP-miRNC (negative control miRNA) were prepared mixing an emulsion of calcium which contained miRNA with a phosphate emulsion, followed by addition of DOPA to make the inner core of LCP. The outer leaflet of LCP contained DOTAP, Cholesterol and DSPE-PEG as. Galactose was conjugated at LCP surface for its targeted delivery to hepatocytes. For induction of hepatic metastasis, HT29-Luc cells were grown on liver biomatrix (BMX) for 7-10 days to acquire metastatic features and then harvested and injected into portal veins of nu/nu mice. Hepatic metastases formation was monitored weekly in vivo using bioluminescent imaging. LCP-miR122 or LCP-miRNC was systematically injected three times a week for 3 weeks starting 24 hours after tumor inoculations. Results: LCP particles were found to be 25-50 nm via dynamic light scattering analysis. When injected in vivo, LCP containing miR-122 was majorly distributed into liver (80%) and was detected in hepatocytes through confocal microscopy and target gene expression analysis. LCP-miR122 did not show any sign of hepatotoxicity or renal toxicity or any other pathological damage to other major organs. The injected conditioned HT29 cells formed metastatic foci within liver 1-3 weeks after portal vein injection as detected by bioluminescent imaging. Interestingly, we found that 67% of (6 out of 9) mice treated with LCP-miR122 did not show any sign of liver metastasis after 3 weeks compared with only 20% (2 out of 10) of mice treated with LCP containing negative control miRNA [OR=0.12, 95% CI:0.015-0.99; p=0.05]. Monitoring liver metastasis expansion on weekly basis, mice treated with LCP-miR122 showed a slight decrease in number of liver metastatic foci compared with control mice which showed a significant increase in number of liver metastatic foci during treatment [0.7 vs 3.8-fold; p=0.05]. Conclusion: These results suggest that targeted delivery of miR-122 into hepatocytes of patients prone to liver metastasis or harboring liver metastasis can prevent or regress hepatic metastasis. Citation Format: Hossein Sendi, Nikhila Sultanpuram, Jie Wang, Amanda Graboski, Liantao Li, Mengying Hu, Leaf Huang, Andrew Z. Wang. Targeted delivery of miR-122 into hepatocytes regresses liver metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5033.

Nano-delivery of Gemcitabine Derivative as a Therapeutic Strategy in a Desmoplastic KRAS Mutant Pancreatic Cancer.

Pancreatic ductal adenocarcinoma remains one of the challenging malignancies to treat, and chemotherapy is the primary treatment strategy available to most patients. Gemcitabine, one of the oldest chemotherapeutic drugs approved for pancreatic cancer, has limited efficacy, due to low drug distribution to the tumor and chemoresistance following therapy. In this study, we delivered gemcitabine monophosphate using lipid calcium phosphate nanoparticles, to desmoplastic pancreatic tumors. Monophosphorylation is a critical, rate-limiting step following cellular uptake of gemcitabine and precursor of the pharmacologically active gemcitabine triphosphate. Our drug delivery strategy enabled us to achieve robust tumor regression with a low parenteral dose in a clinically relevant, KRAS mutant, syngeneic orthotopic allograft, lentivirus-transfected KPC cell line-derived model of pancreatic cancer. Treatment with gemcitabine monophosphate significantly increased apoptosis of cancer cells, enabled reduction in the proportion of immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells, and did not increase expression of cancer stem cell markers. Overall, we could trigger a strong antitumor response in a treatment refractory PDAC model, while bypassing critical hallmarks of gemcitabine chemoresistance.

Sialic acid-modified dexamethasone lipid calcium phosphate gel core nanoparticles for target treatment of kidney injury.

Acute kidney injury (AKI) is a common clinical disease with high morbidity and mortality. Glucocorticoids are drugs that effectively relieve AKI, but the systemic side effects of long-term use limit their use. Herein, we constructed sialic acid-modified dexamethasone sodium phosphate (Dsp)-loaded lipid calcium phosphate gel core nanoparticles (SA-NPs) for the targeted treatment of ischemia-reperfusion (I/R)-induced AKI to improve efficacy and reduce side effects. The obtained nanoparticles could effectively encapsulate Dsp with 66.8% encapsulation efficiency and 4.56% (w/w) drug content. In vitro release indicates that the nanoparticles have a certain sustained release effect and have the characteristics of acid-sensitive release. And SA-NPs significantly increased the cellular uptake and kidney accumulation respectively through the combination of SA and E-selectin receptors overexpressed in inflamed vascular endothelial cells. Besides, the in vivo pharmacokinetic studies showed that Dsp-loaded SA-NPs significantly increased the residence time in the body and their plasma half-life was 1.7 times that of free Dsp. SA-NPs significantly improved the renal function, decreased the level of pro-inflammatory factors, and adjusted the oxidative stress factors and apoptotic proteins compared to free Dsp solution in pharmacodynamic studies. Moreover, little negative effects on blood glucose and bone mineral density were observed. Our study might provide a new strategy for the safe and effective targeting treatment of AKI or other related inflammatory diseases.

Nanoparticle Delivery of RIG-I Agonist Enables Effective and Safe Adjuvant Therapy in Pancreatic Cancer.

Local immunomodulation can be a promising strategy to augment the efficacy and decrease off-target toxicities associated with cancer treatment. Pancreatic cancer is resistant to immunotherapies due to the immunosuppressive tumor microenvironment. Herein, we investigated a therapeutic approach involving delivery of a short interfering double-stranded RNA (dsRNA), specific to Bcl2, with 5' triphosphate ends, by lipid calcium phosphate nanoparticles, in an orthotopic allograft KPC model of pancreatic cancer. Retinoic acid-inducible gene I (RIG-I)-like receptors can bind to 5' triphosphate dsRNA (ppp dsRNA), a pathogen-associated molecular pattern, producing type I interferon, while Bcl2 silencing can drive apoptosis of cancer cells. Our approach demonstrated a robust enrichment of tumor tissue with therapeutic nanoparticles and enabled a significant tumor growth inhibition, prolonging median overall survival. Nanoparticles encapsulating dual-therapeutic ppp dsRNA allowed strong induction in levels of pro-inflammatory Th1 cytokines, further increasing proportions of CD8+ Tcells over regulatory Tcells, M1 over M2 macrophages, and decreased levels of immunosuppressive B regulatory and plasma cells in the tumor microenvironment. Thus, these results provide a new immunotherapy approach for pancreatic cancer.

Co-Delivery of Gemcitabine and Paclitaxel in cRGD-Modified Long Circulating Nanoparticles with Asymmetric Lipid Layers for Breast Cancer Treatment

Combination chemotherapy is a common clinical practice in cancer treatment. Here, cyclic RGD (arginylglycylaspartic acid) peptide was introduced to the surface of lipid/calcium/phosphate (LCP) asymmetric lipid layer nanoparticles for the co-delivery of paclitaxel (PTX) and gemcitabine monophosphate (GMP) (P/G-NPs). The sphere-like morphology of P/G-NPs displays a well-distributed particle size, and high entrapment efficiency and drug loading for both PTX and GMP, with a positive zeta potential. P/G-NPs were stable for up to 15 days. The cellular uptake of these cyclic RGD-modified nanoparticles was significantly higher than that of unmodified nanoparticles over 2 h incubation. Compared with the combination of free PTX and GMP (P/G-Free), P/G-NPs exhibited a longer circulation lifetime and improved absorption for PTX and GMP. Polyethylene glycol was responsible for a higher plasma concentration and a decreased apparent volume of distribution (Vz). Nanoparticles enhanced the drug accumulation in tumors compared with other major organs after 24 h. P/G-NPs nearly halted tumor growth, with little evidence of general toxicity, whereas P/G-Free had only a modest inhibitory effect at 16 mg/kg of GMP and 2.0 mg/kg of PTX. Increased levels of apoptosis within tumors were detected in P/G-NPs group by approximately 43.6% (TUNEL assay). When compared with GMP NPs, PTX NPs, and P/G-Free, P/G-NPs decreased expression of B-cell lymphoma-2 and B-cell lymphoma-extra large proteins, and increased expression of cleaved poly-ADP-ribose polymerase-1. Calreticulin expression in tumors also increased upon the co-delivery of PTX and GMP. The antitumor effect of P/G-NPs is more powerful than P/G-Free, GMP NP, or PTX NP alone, without obvious toxicity.

Liver specific gene immunotherapies resolve immune suppressive ectopic lymphoid structures of liver metastases and prolong survival

Our approach results in transient liver specific expression of gene immunotherapies with improved efficacy and reduced off-target toxicities over traditional systemically administered immunotherapies. This approach would allow clinicians to manipulate the liver and immune microenvironment to resist cancer invasion, improve organ health, and prolong patient survival.

Investigation of phosphorylated adjuvants co-encapsulated with a model cancer peptide antigen for the treatment of colorectal cancer and liver metastasis

The lipid calcium phosphate nanoparticle is a versatile platform capable of encapsulating a wide range of phosphorylated molecules from single nucleotides to pDNA. The use of this platform has shown great success as an immunotherapeutic vaccine carrier, capable of delivering co-encapsulated phosphorylated adjuvants and peptides. Three potent vaccine formulations were investigated for anti-cancer efficacy. The phosphorylated adjuvants, CpG, 2′3′cGAMP, and 5′pppdsRNA were co-encapsulated with a model phosphorylated tumor specific peptide antigen (p-AH1-A5). The anti-cancer efficacy of these adjuvants was assessed using an orthotopic colorectal liver metastasis model based on highly aggressive and metastatic CT-26 FL3 cells implanted into the cecum wall. The results clearly indicate that the RIG-1 ligand, 5′pppdsRNA, co-encapsulated with the p-AH1-A5 peptide antigen greatly reduced the growth rate of the primary colon cancer as well as arrested the establishment of liver metastasis in comparison to the other adjuvant formulations and unvaccinated controls. Further evaluation of the immune cell populations within the primary tumor confirms the ability of the 5′pppdsRNA adjuvant to boost the adaptive CD8+ T-cell population, while not inciting increased populations of immune suppressive cell types such as T-regulatory cells or myeloid derived suppressor cells. Furthermore, to our knowledge this is the first study to investigate the anti-cancer efficacy of a specific RIG-1 receptor ligand, 5′pppdsRNA, alongside more established TLR 9 (CpG) and STING (2′3′cGAMP) adjuvants in a cancer vaccine. The 5′pppdsRNA vaccine formulation can be a potent immunotherapy, especially when combined with agents that remodel the immune suppressive microenvironment of the tumor.

Exosomes from M1-Polarized Macrophages Potentiate the Cancer Vaccine by Creating a Pro-inflammatory Microenvironment in the Lymph Node

Exosomes are small membrane-bound vesicular particles generated by most cells for intercellular communication and regulation. During biogenesis, specific lipids, RNAs, proteins, and carbohydrates are enriched and packaged into the vesicles so that the exosomal contents reflect not only the source but also the physiological conditions of the parental cells. These exosomes transport materials or signals to the target cells for diverse physiological purposes. Our study focused on the exosomes derived from M1-polarized, proinflammatory macrophages for the possibility of using M1 exosomes as an immunopotentiator for a cancer vaccine. The M1 exosomes displayed a tropism toward lymph nodes after subcutaneous injection, primarily taken up by the local macrophages and dendritic cells, and they induced the release of a pool of Th1 cytokines. We found that M1, but not M2, exosomes enhanced activity of lipid calcium phosphate (LCP) nanoparticle-encapsulated Trp2 vaccine, and they induced a stronger antigen-specific cytotoxic Tcell response. The M1 exosomes proved to be a more potent immunopotentiator than CpG oligonucleotide when used with LCP nanoparticle vaccine in a melanoma growth inhibition study. Thus, our study indicated that exosomes derived from M1-polarized macrophages could be used as a vaccine adjuvant.

Tumor-targeted delivery of sunitinib base enhances vaccine therapy for advanced melanoma by remodeling the tumor microenvironment

Development of an effective treatment against advanced tumors remains a major challenge for cancer immunotherapy. We have previously developed a potent mannose-modified lipid calcium phosphate (LCP) nanoparticle (NP)-based Trp2 vaccine for melanoma therapy, but because this vaccine can induce a potent anti-tumor immune response only during the early stages of melanoma, poor tumor growth inhibition has been observed in more advanced melanoma models, likely due to the development of an immune-suppressive tumor microenvironment (TME). To effectively treat this aggressive tumor, a multi-target receptor tyrosine kinase inhibitor, sunitinib base, was efficiently encapsulated into a targeted polymeric micelle nano-delivery system (SUNb-PM), working in a synergistic manner with vaccine therapy in an advanced mouse melanoma model. SUNb-PM not only increased cytotoxic T-cell infiltration and decreased the number and percentage of MDSCs and Tregs in the TME, but also induced a shift in cytokine expression from Th2 to Th1 type while remodeling the tumor-associated fibroblasts, collagen, and blood vessels in the tumor. Additionally, inhibition of the Stat3 and AKT signaling pathways by SUNb-PM may induce tumor cell apoptosis or decrease tumor immune evasion. Our findings indicated that targeted delivery of a tyrosine kinase inhibitor to tumors can be used in a novel synergistic way to enhance the therapeutic efficacy of existing immune-based therapies for advanced melanoma.

Current and Future Theranostic Applications of the Lipid-Calcium-Phosphate Nanoparticle Platform.

Over the last four years, the Lipid-Calcium-Phosphate (LCP) nanoparticle platform has shown success in a wide range of treatment strategies, recently including theranostics. The high specific drug loading of radiometals into LCP, coupled with its ability to efficiently encapsulate many types of cytotoxic agents, allows a broad range of theranostic applications, many of which are yet unexplored. In addition to providing an overview of current medical imaging modalities, this review highlights the current theranostic applications for LCP using SPECT and PET, and discusses potential future uses of the platform by comparing it with both systemically and locally delivered clinical radiotherapy options as well as introducing its applications as an MRI contrast agent. Strengths and weaknesses of LCP and of nanoparticles in general are discussed, as well as caveats regarding the use of fluorescence to determine the accumulation or biodistribution of a probe.

A radio-theranostic nanoparticle with high specific drug loading for cancer therapy and imaging

We have developed a theranostic nanoparticle delivering the model radionuclide 177Lu based on the versatile lipid-calcium-phosphate (LCP) nanoparticle delivery platform. Characterization of 177Lu-LCP has shown that radionuclide loading can be increased by several orders of magnitude without affecting the encapsulation efficiency or the morphology of 177Lu-LCP, allowing consistency during fabrication and overcoming scale-up barriers typical of nanotherapeutics. The choice of 177Lu as a model radionuclide has allowed in vivo anticancer therapy in addition to radiographic imaging via the dual decay modes of 177Lu. Tumor accumulation of 177Lu-LCP was measured using both SPECT and Cerenkov imaging modalities in live mice, and treatment with just one dose of 177Lu-LCP showed significant in vivo tumor inhibition in two subcutaneous xenograft tumor models. Microenvironment and cytotoxicity studies suggest that 177Lu-LCP inhibits tumor growth by causing apoptotic cell death via double-stranded DNA breaks while causing a remodeling of the tumor microenvironment to a more disordered and less malignant phenotype.

Nanoparticle delivery of HIF1α siRNA combined with photodynamic therapy as a potential treatment strategy for head-and-neck cancer

Combination therapy has become a major strategy in cancer treatment. We used anisamide-targeted lipid–calcium–phosphate (LCP) nanoparticles to efficiently deliver HIF1α siRNA to the cytoplasm of sigma receptor-expressing SCC4 and SAS cells that were also subjected to photodynamic therapy (PDT). HIF1α siRNA nanoparticles effectively reduced HIF1α expression, increased cell death, and significantly inhibited cell growth following photosan-mediated photodynamic therapy in cultured cells. Intravenous injection of the same nanoparticles into human SCC4 or SAS xenografted mice likewise resulted in concentrated siRNA accumulation and reduced HIF1α expression in tumor tissues. When combined with photodynamic therapy, HIF1α siRNA nanoparticles enhanced the regression in tumor size resulting in a ~40% decrease in volume after 10 days. Combination therapy was found to be substantially more effective than either HIF1α siRNA or photodynamic therapy alone. Results from caspase-3, TUNEL, and CD31 marker studies support this conclusion. Our results show the potential use of LCP nanoparticles for efficient delivery of HIF1α siRNA into tumors as part of combination therapy along with PDT in the treatment of oral squamous cell carcinoma.

Nanoparticle-Delivered Transforming Growth Factor-β siRNA Enhances Vaccination against Advanced Melanoma by Modifying Tumor Microenvironment

Achievement of potent immunoresponses against self/tumor antigens and effective therapeutic outcome against advanced tumors remain major challenges in cancer immunotherapy. The specificity and efficiency of two nanoparticle-based delivery systems, lipid-calcium-phosphate (LCP) nanoparticle (NP) and liposome-protamine-hyaluronic acid (LPH) NP, provide us an opportunity to address both challenges. A mannose-modified LCP NP delivered both tumor antigen (Trp 2 peptide) and adjuvant (CpG oligonucleotide) to the dendritic cells and elicited a potent, systemic immune response regardless of the existence or the stage of tumors in the host. This vaccine was less effective, however, against later stage B16F10 melanoma in a subcutaneous syngeneic model. Mechanistic follow-up studies suggest that elevated levels of immune-suppressive cytokines within the tumor microenvironment, such as TGF-β, might be responsible. We strategically augment the efficacy of LCP vaccine on an advanced tumor by silencing TGF-β in tumor cells. The delivery of siRNA using LPH NP resulted in about 50% knockdown of TGF-β in the late stage tumor microenvironment. TGF-β down-regulation boosted the vaccine efficacy and inhibited tumor growth by 52% compared with vaccine treatment alone, as a result of increased levels of tumor infiltrating CD8+ T cells and decreased level of regulatory T cells. Combination of systemic induction of antigen-specific immune response with LCP vaccine and targeted modification of tumor microenvironment with LPH NP offers a flexible and powerful platform for both mechanism study and immunotherapeutic strategy development.

Lipid–calcium phosphate nanoparticles for delivery to the lymphatic system and SPECT/CT imaging of lymph node metastases

A lipid/calcium/phosphate (LCP) nanoparticle (NP) formulation (particle diameter ∼25 nm) with superior siRNA delivery efficiency was developed and reported previously. Here, we describe the successful formulation of 111In into LCP for SPECT/CT imaging. Imaging and biodistribution studies showed that, polyethylene glycol grafted 111In-LCP preferentially accumulated in the lymph nodes at ∼70% ID/g in both C57BL/6 and nude mice when the improved surface coating method was used. Both the liver and spleen accumulated only ∼25% ID/g. Larger LCP (diameter ∼67 nm) was less lymphotropic. These results indicate that 25 nm LCP was able to penetrate into tissues, enter the lymphatic system, and accumulate in the lymph nodes via lymphatic drainage due to 1) small size, 2) a well-PEGylated lipid surface, and 3) a slightly negative surface charge. The capability of intravenously injected 111In-LCP to visualize an enlarged, tumor-loaded sentinel lymph node was demonstrated using a 4T1 breast cancer lymph node metastasis model. Systemic gene delivery to the lymph nodes after IV injection was demonstrated by the expression of red fluorescent protein cDNA. The potential of using LCP for lymphatic drug delivery is discussed.