Lipid-coated Calcium Phosphate Research Articles

Disulfiram loaded calcium phosphate nanoparticles for enhanced cancer immunotherapy

Considering the huge cost and long test periods required for new drug development, repurposing drugs that have already been applied in the clinic as new cancer treatment candidates represents an attractive alternative. Disulfiram (DSF) was originally used to treat alcoholism and has proven to have anticancer effects with the coadministration of copper ions (Cu2+). However, the limited water-solubility of DSF and systemic toxicity induced by exogenous Cu2+ hinder its practical application. Herein, we constructed pH-responsive lipid-coated calcium phosphate nanoparticles (LCP NPs) co-loaded with Cu2+ and DSF. After intravenous injection, those nanoparticles with long blood half-life preferentially accumulate in tumors, followed by the degradation of nanoparticles in response to the acidic tumor microenvironment, subsequently releasing Cu2+ and DSF to generate cytotoxic metabolite DTC−Copper complex, bis(diethyldithiocarbamate)–copper (CuET) for tumor treatment. In addition to direct cytotoxicity, the active metabolite CuET could effectively induce immunogenic cell death (ICD) of cancer cells to regulate the immunosuppressive tumor microenvironment, contributing to enhanced immune checkpoint blockade (ICB) therapy in triggering systemic immune responses. This work thus demonstrates the great promises of repurposing the old drug DSF as a new ICD inducer with nano-formulation, to achieve improved synergetic tumor-responsive therapy with low side effects.

PD-L1-Targeted Co-Delivery of Two Chemotherapeutics for Efficient Suppression of Skin Cancer Growth.

To overcome the severe side effects of cancer chemotherapy, it is vital to develop targeting chemotherapeutic delivery systems with the potent inhibition of tumour growth, angiogenesis, invasion and migration at low drug dosages. For this purpose, we co-loaded a conventional antiworm drug, albendazole (ABZ), and a TOPK inhibitor, OTS964, into lipid-coated calcium phosphate (LCP) nanoparticles for skin cancer treatment. OTS- and ABZ-loaded LCP (OTS-ABZ-LCP) showed a synergistic cytotoxicity against skin cancer cells through their specific cancerous pathways, without obvious toxicity to healthy cell lines. Moreover, dual-targeting the programmed death ligand-1 (PD-L1) and folate receptor overexpressed on the surface of skin cancer cells completely suppressed the skin tumour growth at low doses of ABZ and OTS. In summary, ABZ and OTS co-loaded dual-targeting LCP NPs represent a promising platform with high potentials against complicated cancers where PD-L1/FA dual targeting appears as an effective approach for efficient and selective cancer therapy.

Oridonin-loaded lipid-coated calcium phosphate nanoparticles: preparation, characterization, and application in A549 lung cancer

In this study, the feasibility of using the novel anisamide-lipid calcium phosphate nanoparticles (AS-LCPs) as a nanocarrier for the delivery of biologically active oridonin (ORD) was evaluated on lung cancer models. In addition to the characterization, release behaviors, and stability of AS-ORD LCPs, we also studied their pharmacokinetics and targeting ability by in vivo imaging. Finally, we investigated the effect of ORD on the anti-tumor efficiency of the AS-LCPs based on weight, tumor inhibition, and the survival time of mice. The average particle size of the AS-ORD LCPs was 129.5 ± 23.7 nm, the polydispersity index (PDI) was 0.16 ± 0.03, and the zeta potential was 23.6 ± 3.4 mV. The AS-ORD LCPs were proved to be stable under both long-term and accelerated storage conditions. The AS-ORD LCPs showed sustained release in vivo and faster release in acidic environment, which was favorable to drug release in tumor environment. In vivo studies showed that depending on surface modification, AS-ORD LCPs which suggested that cell internalization was change and more drugs entered the cells successfully. Therefore, AS-ORD LCPs could be a promising formulation for the treatment of lung cancer.

Encapsulating Anti-Parasite Benzimidazole Drugs into Lipid-Coated Calcium Phosphate Nanoparticles to Efficiently Induce Skin Cancer Cell Apoptosis

Benzimidazole (BMZ) family of anti-worm drugs has been now repurposed as anti-cancer drugs. However, offering a general reformulation method for these drugs is essential due to their hydrophobicity and low aqueous solubility. In this work, we developed a general approach to load typical BMZ drugs as tiny nanocrystals within lipid-coated calcium phosphate (LCP) nanoparticles. BMZ drug-loaded LCP nanoparticles increased their solubility in PBS by 100–200% and significantly enhanced the anti-cancer efficacy in the treatment of B16F0 melanoma cells. These drug-LCP nanoparticles induced much more cancer cell apoptosis, generated much more reactive oxygen species (ROS) and inhibited Bcl-2 expression of cancer cells. Moreover, BMZ drug-loaded LCP nanoparticles caused morphological change and extension disruption of cancer cells, and significantly reduced migration activity, representing high possibility for inhibition of tumor dissemination and metastasis. Very advantageously, BMZ drug-loaded LCP nanoparticles did not show any obvious toxicity, Bcl-2 inhibition and morphological changes in HEK293T healthy cells. In conclusion, BMZ drug-incorporated LCP nanoformulations may be a valuable nanomedicine that is able to inhibit primary tumors and prevent tumor dissemination with minimum side effects on healthy cells and tissues.

Nanostructuring a Widely Used Antiworm Drug into the Lipid-Coated Calcium Phosphate Matrix for Enhanced Skin Tumor Treatment.

Recently, a widely used antiworm drug albendazole (ABZ) has been recognized as an anticancer drug, while being safe for healthy cells. However, its clinical application is limited due to low water solubility. In this work, we incorporated ABZ into pH-responsive lipid-coated calcium phosphate (LCP) nanoparticles to enhance anticancer efficacy. LCP-ABZ nanoparticles doubled the ABZ solubility with a much quicker release rate in the slightly acidic environment. LCP-ABZ significantly improved cytotoxicity to B16F0 cells and reduced their migration probably through enhanced apoptosis induction via reactive oxygen species overproduction. In a B16F0-bearing mouse models, LCP-ABZ nanoparticles reduced the tumor size by 50-60% at the total dose of only 15 mg (ABZ)/kg, more effective than commercial albendazole and other ABZ nanoparticles. Overall, this research demonstrated LCP nanoparticle as an effective platform to deliver ABZ for cancer therapy, with safety to healthy tissues and the potential to inhibit metastasis.

<p>Doxorubicin-Loaded Carbon Dots Lipid-Coated Calcium Phosphate Nanoparticles for Visual Targeted Delivery and Therapy of Tumor</p>

BackgroundCarbon dots (CDs) have attracted extensive attention in recent years because of their high biocompatibility and unique optical property. But they could not be well applied in the drug delivery system to enable distribution in tumor sites with their low pH sensitivity. They are barriers for drug delivery. CDs as an imaging proper were conjugated with doxorubicin (DOX) lipid-coated calcium phosphate (LCP) nanoparticle, for a pH-sensitive nanocarrier and delivery of the antitumor drugs.Materials and MethodsCDs were prepared by one-step hydrothermal treatment of citric acid and ethylenediamine. The nanoparticles were simply prepared by using microemulsion technology to form calcium phosphate (CaP) core and further coated with cationic lipids.ResultsThe structure was characterized by FTIR, XRD and TEM. In vitro release study revealed that DOX-CDs@LCP was pH dependent. The cytotoxicity assay demonstrated that it exhibited enhanced efficiency compared to the control group (DOX-CDs), but weaker than free DOX. The cellular uptake revealed that these pH-sensitive nanoparticles could be taken up effectively and deliver DOX into the cytoplasm to reach antitumor effect. The fluorescence imaging indicated that DOX-CDs@LCP mostly distributed in the tumor region due to the enhanced permeability and retention effect (EPR) to reduce its systematical toxicity. Importantly, an antitumor activity study demonstrated that the DOX-CDs@LCP nanoparticles had higher antitumor activity than any other groups and lower toxicity. The results showed that LCP could significantly promote the release in tumor microenvironment due to pH-response. The DOX-CDs could enhance load capacity and reduce drug premature releasing; real-time tracking of efficacy as confocal imaging contrast agent. Thus, DOX-CDs@LCP had antitumor capacity and lower systematic toxicity in tumor therapy.ConclusionDOX-CDs@LCP were proven as a promising tumor pH-sensitive and imaging-guided drug delivery system for liver cancer chemotherapy.

Boosting the Ferroptotic Antitumor Efficacy via Site-Specific Amplification of Tailored Lipid Peroxidation.

Ferroptosis is an iron-dependent cell death pathway that can eradicate certain apoptosis-insensitive cancer cells. The ferroptosis-inducing molecules are tailored lipid peroxides whose efficacy is compromised in hypoxic solid tumor and lack of tumor selectivity. It has been demonstrated that ascorbate (Asc) in pharmacological concentrations can selectively kill cancer cells via accumulating hydrogen peroxide (H2O2) only in tumor extracellular fluids. It was hypothesized that Asc-induced, selective enrichment of H2O2 in tumor coupled with Fe3+ codelivery could simultaneously address the above two problems via boosting the levels of hydroxyl radicals and oxygen in the tumor site to ease peroxidation initiation and propagation, respectively. The aim of this work was to synergize the action of Asc with lipid-coated calcium phosphate (CaP) hybrid nanocarrier that can concurrently load polar Fe3+ and nonpolar RSL3, a ferroptosis inducer with the mechanism of inhibiting lipid peroxide repair enzyme (GPX4). The hybrid nanocarriers showed accelerated cargo release at acidic conditions (pH 5.0). The combinational approach (Asc plus nanocarrier) produced significantly elevated levels of hydroxyl radicals, lipid peroxides, and depleted glutathione under hypoxia, which was accompanied with the strong cytotoxicity (IC50 = 1.2 ± 0.2 μM) in the model 4 T1 cells. In the 4 T1 tumor-bearing xenograft mouse model, the intravenous nanocarrier delivery plus intraperitoneal Asc administration resulted in a superior antitumor performance in terms of tumor suppression, which did not produce supplementary adverse effects to the healthy organs. This work provides a novel approach to enhance the potency of ferroptotic nanomedicine against solid tumors without inducing additional side effects.

Multifunctional lipid-coated calcium phosphate nanoplatforms for complete inhibition of large triple negative breast cancer via targeted combined therapy

Combined and targeted therapy have been extensively employed to achieve more effective elimination of tumor tissues. In this study, biocompatible multifunctional lipid-coated calcium phosphate nanoparticles (LCP NPs) were designed and constructed as an efficient targeted delivery system for combined gene/photothermal therapy to inhibit growth of the triple negative breast tumor (MDA-MB-468) in vitro and in vivo. LCP NPs were functionalized with a bispecific antibody (BsAb) via non-covalent bond specific for methoxy group of PEG (mPEG) on the particle surface. This BsAb is also able to target epidermal growth factor receptor (EGFR) expressed on MDA-MB-468 cells. Such LCP-BsAb NPs loaded with Cell Death (CD)-siRNA and indocyanine green (ICG) were efficiently taken up by MDA-MB-468 cells, significantly inducing cell apoptosis and synergistically suppressing cell proliferation upon irradiation of 808 nm near-infrared laser. These targeted multifunctional LCP NPs more efficiently accumulated in the tumor tissue. The combined RNAi (CD-siRNA) and photothermal (ICG) therapy using the targeted LCP NPs nearly eliminated both small tumors (∼100 mm3) and large tumors (∼500 mm3) in the mouse model. Thus, the well-devised multifunctional LCP NPs are one of the most promising delivery systems for combined and targeted cancer therapy.

Sequential Targeting TGF‐β Signaling and KRAS Mutation Increases Therapeutic Efficacy in Pancreatic Cancer

Pancreatic cancer is a highly aggressive malignancy that strongly resists extant treatments. The failure of existing therapies is majorly attributed to the tough tumor microenvironment (TME) limiting drug access and the undruggable targets of tumor cells. The formation of suppressive TME is regulated by transforming growth factor beta (TGF-β) signaling, while the poor response and short survival of almost 90% of pancreatic cancer patients results from the oncogenic KRAS mutation. Hence, simultaneously targeting both the TGF-β and KRAS pathways might dismantle the obstacles of pancreatic cancer therapy. Here, a novel sequential-targeting strategy is developed, in which antifibrotic fraxinellone-loaded CGKRK-modified nanoparticles (Frax-NP-CGKRK) are constructed to regulate TGF-β signaling and siRNA-loaded lipid-coated calcium phosphate (LCP) biomimetic nanoparticles (siKras-LCP-ApoE3) are applied to interfere with the oncogenic KRAS. Frax-NP-CGKRK successfully targets the tumor sites through the recognition of overexpressed heparan sulfate proteoglycan, reverses the activated cancer-associated fibroblasts (CAFs), attenuates the dense stroma barrier, and enhances tumor blood perfusion. Afterward, siKras-LCP-ApoE3 is efficiently internalized by the tumor cells through macropinocytosis and specifically silencing KRAS mutation. Compared with gemcitabine, this sequential-targeting strategy significantly elongates the lifespans of pancreatic tumor-bearing animals, hence providing a promising approach for pancreatic cancer therapy.

Silencing PD-1 and PD-L1 with nanoparticle-delivered small interfering RNA increases cytotoxicity of tumor-infiltrating lymphocytes.

To determine if silencing PD-1 on tumor-infiltrating lymphocytes (TILs) and its ligand-1 (PD-L1) on cancer cells will enhance the cytotoxicity of TILs. Lipid-coated calcium phosphate nanoparticles were synthesized to deliver siRNAs against PD-1 and PD-L1 to TILs and breast cancer MCF-7 cells. The downregulation of PD-1/PD-L1 expressions was determined by real-time PCR and western blotting assays. The killing efficacy of TILs to MCF-7 cells was determined by cytotoxic T lymphocyte assay. Lipid-coated calcium phosphate nanoparticles effectively delivered siRNAs and silenced PD-1 and PD-L1sh expression. The knockdown of either gene or both greatly improved the cytotoxicity of TILs. Silencing PD-1 and PD-L1 is an effective approach to increase TIL cytotoxicity to cancer cells.

Enhancing PD-1 Gene Silence in T Lymphocytes by Comparing the Delivery Performance of Two Inorganic Nanoparticle Platforms.

Suitable carriers are crucial to RNAi applications for cancer genotherapy and T-cell immunotherapy. In this research, we selected two extensively-investigated biocompatible inorganic nanoparticle carriers, i.e., layered double hydroxide (LDH) and lipid-coated calcium phosphate (LCP) and then compared their efficacy for siRNA delivery in T cells, in order to understand which carrier is more efficient in delivering functional programmed cell death protein 1 siRNA (PD-1 siRNA) to suspended T lymphocytes. Both LDH and LCP nanoparticles quickly delivered gene segment to mouse T cell lines (EL4), while the LCP nanoparticles exhibited more cellular uptake and higher PD-1 gene silence efficiency. We further demonstrated that LCP nanoparticles successfully reduced the expression of PD-1 in human ex vivo tumor infiltrating lymphocytes (TILs). Thus, LCP nanoparticles can be used as a better nano-carrier for gene therapy in lymphocytes, especially in regards to TIL-related cancer immunotherapy.

Gemcitabine nanoparticles promote antitumor immunity against melanoma

Myeloid-derived suppressor cells (MDSCs) promote tumor-mediated immunosuppression and cancer progression. Gemcitabine (Gem) is a MDSC-depleting chemotherapeutic agent; however, its clinical use is hampered by its drug resistance and inefficient in vivo delivery. Here we describe a strategy to formulate a Gem analogue gemcitabine monophosphate (GMP) into a lipid-coated calcium phosphate (LCP) nanoparticle, and investigate its antitumor immunity and therapeutic effects after systemic administrations. In the syngeneic mouse model of B16F10 melanoma, compared with free Gem, the LCP-formulated GMP (LCP-GMP) significantly induced apoptosis and reduced immunosuppression in the tumor microenvironment (TME). LCP-GMP effectively depleted MDSCs and regulatory T cells, and skewed macrophage polarization towards the antitumor M1 phenotype in the TME, leading to enhanced CD8+ T-cell immune response and profound tumor growth inhibition. Thus, engineering the in vivo delivery of MDSC-depleting agents using nanotechnology could substantially modulate immunosuppressive TME and boost T-cell immune response for enhanced antitumor efficacy.

Enhanced delivery of siRNA to triple negative breast cancer cells in vitro and in vivo through functionalizing lipid-coated calcium phosphate nanoparticles with dual target ligands.

The conjugation of ligands to nanoparticle platforms for the target delivery of therapeutic agents to the tumor tissue is one of the promising anti-cancer strategies. However, conventional nanoparticle platforms are not so effective in terms of the selectivity and transfection efficiency. In this study, we designed and developed a dual-target drug/gene delivery system based on lipid-coated calcium phosphate (LCP) nanoparticles (NPs) for significantly enhanced siRNA cellular uptake and transfection efficiency. LCP NPs loaded with therapeutic siRNA were conjugated with a controlled number of folic acid and/or EGFR-specific single chain fragment antibody (ABX-EGF scFv). The uptake of ABX-EGF scFv-modified (LCP-scFv) and folic acid-modified LCP NPs (LCP-FA) by human breast tumor cells (MDA-MB-468) was significantly higher with an optimal ligand density on each NP surface (LCP-125scFv and LCP-100FA). Co-conjugation with sub-optimal dual ligands (50 FA and 75 ABX-EGF scFv) per LCP NP (LCP-50FA-75scFv) further enhanced the cellular uptake. More significantly, much more NPs were delivered to the MDA-MB-468 tumor tissue in the nude mouse model when LCP-50FA-75scFv NPs were used. Therefore, the new dual-ligand LCP NPs may be a valuable targeting system for human breast cancer diagnosis and therapy.

MRNA Vaccine with Antigen-Specific Checkpoint Blockade Induces an Enhanced Immune Response against Established Melanoma.

We reported a preclinical cancer vaccine that simultaneously introduced an mRNA antigen and an immune checkpoint blocking siRNA into the antigen-presenting cells. This was achieved by formulating both nucleic acid-based immunotherapeutics into a lipid-coated calcium phosphate (LCP) nanoparticle (NP) as a carrier to address the delivery challenge. The PEGylated lipid NPs were functionalized with mannose as thetargeting ligand to facilitate the preferential uptake by the dendritic cells (DCs) in the lymph nodes after subcutaneous administration. The calcium phosphate core allowed acid-mediated dissolution in the endo-lysosomal compartment, which prompted rapid release of cargoes after cellular internalization of NP. LCP mRNA vaccine encoding TRP2 elicited a robust antigen-specific cytotoxic Tcell response and a humoral immune response in a C57BL/6 mouse model of B16F10 melanoma. The immune responses efficaciously inhibited the melanoma growth. Moreover, co-delivery of PD-L1 siRNA and mRNA vaccine resulted in the downregulation of PD-L1 in the DCs that presented tumor antigens, significantly prompting Tcell activation and proliferation. The enhanced Tcell response had a profound inhibitory effect on tumor growth and metastasis. Generally, the work provided a paradigm for the development of an mRNA vaccine carrier to boost the anticancer immune response.

BRAF peptide vaccine facilitates therapy of murine BRAF-mutant melanoma.

Approximately, 50% of human melanomas are driven by BRAF mutations, which produce tumors that are highly immunosuppressive and often resistant to vaccine therapy. We introduced lipid-coated calcium phosphate nanoparticles (LCP NPs) as a carrier to efficiently deliver a tumor-specific antigen, the BRAFV600E peptide, to drive dendritic cell (DC) maturation and antigen presentation in C57BL6 mice. The BRAF peptide vaccine elicited a robust, antigen-specific cytotoxic T cell response and potent tumor growth inhibition in a murine BRAF-mutant melanoma model. Advanced BRAF-specific immune response was illustrated by IFN-γ production assay and cytotoxic T lymphocyte (CTL) assay. Remodeling of immunosuppressive modules within the tumor microenvironment further facilitated CTL infiltration. Thus, using LCP NPs to deliver the BRAF peptide vaccine is a promising strategy for the BRAF-mutant melanoma therapy.

Nanovaccines for remodeling the suppressive tumor microenvironment: New horizons in cancer immunotherapy

Despite limited successes in clinical development, therapeutic cancer vaccines have experienced resurgence in recent years due to an enhanced emphasis upon co-mitigating factors underlying immune response. Specifically, reversing the immune-suppressive effects of the tumor microenvironment, mediated by a variety of cellular and molecular signaling mechanisms, has become fundamental toward enhancing therapeutic efficacy. Therein, our lab has implemented various nano-vaccines based on the lipid-coated calcium phosphate platform for combined immunotherapy, in which antigenic, epitopeassociated peptides as well as immune-suppression inhibitors can be co-delivered, often functioning through the same formulation. In probing the mechanism of action of such systems in vitro and in vivo, an improved effect synergy can be elucidated, inspiring future preclinical efforts and hope for clinical success. Open image in new window

Devising new lipid-coated calcium phosphate/carbonate hybrid nanoparticles for controlled release in endosomes for efficient gene delivery.

Lipid-coated calcium phosphate (LCP) nanoparticles (NPs) are proven to be effective vehicles for the delivery of genes and some drugs, while it is not desirable for NPs to release genes/drugs in late endosomes/lysosomes. To achieve the early endosomal release and escape, we have designed and developed new lipid-coated calcium carbonate/phosphate (LCCP) hybrid NPs. These new hybrid LCCP NPs have a spherical structure with an average diameter of 40 nm and high gene loading capacity. We particularly demonstrate that the loaded dsDNA/siRNA is mostly released under mildly acidic conditions (pH 6.0-5.5). LCCP NPs are also effectively internalized by B16F10 cells in a dose and time dependent way. The delivery efficacy has been further demonstrated using two functional siRNAs, i.e. programmed death ligand 1 (PD-L1) siRNA for PD-L1 silencing and polo-like kinase 1 (PLK1) siRNA for growth inhibition of B16F10. Consistently, the LCCP loaded PD-L1 siRNA shows quicker PD-L1-mRNA inhibition than LCP NPs, indicating that LCCP NPs improved the siRNA release in endosomes.

Maximizing the Supported Bilayer Phenomenon: Liposomes Comprised Exclusively of PEGylated Phospholipids for Enhanced Systemic and Lymphatic Delivery.

Traditional liposomes degrade into lower-order micelles when PEGylated to even minor degrees (6-7 mol %) and therefore can offer only limited steric exclusion against opsonization during in vivo delivery. In this work, we present for the first time a liposome coated exclusively by PEGylated phospholipids, utilizing lipid-coated calcium phosphate (CaP) cores of diverse sizes (10-15 nm, 30-40 nm) as well as varying polyethylene glycol (PEG) chain lengths (350-5000 Da). Such fully-PEGylated liposome calcium phosphate (LCP) particles exhibit a PEG chain length-dependent circulation longevity and robust immune evasion, while facilitating both strong accumulation within solid tumors upon intravenous injection and a more rapid and extensive lymphatic drainage upon subcutaneous administration. Further, these fully-PEGylated liposomes remain amenable to active targeting strategies which facilitate improved degrees of focused distribution and nanoparticle uptake, represent a lipid packing density commensurate with the formation of a lipid bilayer, and avoid use of scale-limited physical resuspension methods. We expect such improved delivery properties to translate into improved therapeutic safety and efficacy for a variety of systemic and lymphatic diseases.

Preparation of optimized lipid-coated calcium phosphate nanoparticles for enhanced in vitro gene delivery to breast cancer cells.

Lipid coated calcium phosphate (LCP) nanoparticles (NPs) remain an attractive option for siRNA systemic delivery. Previous research has shown that the stoichiometry of reactants affects the size and morphology of nanostructured calcium phosphate (CaP) particles. However, it is unclear how synthesis parameters such as the Ca/P molar ratio and mixing style influence the siRNA loading and protection by LCP NPs, and subsequent siRNA delivery efficiency. In this research, we found that the Ca/P molar ratio is critical in controlling the size, zeta potential, dispersion state, siRNA loading and protection. Based on the siRNA loading efficiency and capacity as well as siRNA protection effectiveness, we suggested an optimized LCP NPs delivery system. The optimized LCP NPs had a hollow, spherical structure with the average particle size of ~40 nm and were able to maintain their stability in serum containing media and PBS for over 24 h, with a pH-sensitive dissolution property. The superior ability of optimized LCP NPs to maintain the integrity of encapsulated siRNA and the colloidal stability in culture medium allow this formulation to achieve improved cellular accumulation of siRNA and enhanced growth inhibition of human breast cancer cells in vitro, compared with the commercial transfection agent Oligofectamine™.

Preparation of optimized lipid-coated calcium phosphate nanoparticles for enhanced in vitro gene delivery to breast cancer cells

Preparation of optimized lipid-coated calcium phosphate nanoparticles for enhanced in vitro gene delivery to breast cancer cells