Retarding breast tumor growth with nanoparticle-facilitated intravenous delivery of BRCA1 and BRCA2 tumor suppressor genes.

Gene augmentation therapy entails replacement of the abnormal tumor suppressor genes in cancer cells. In this study, we performed gene augmentation for BRCA1/2 tumor suppressors in order to retard tumor development in breast cancer mouse model. We formulated inorganic carbonate apatite (CA) nanoparticles (NPs) to carry and deliver the purified BRCA1/2 gene- bearing plasmid DNA both in vitro and in vivo. The outcome of BRCA1/2 plasmid-loaded NPs delivery on cellular viability of three breast cancer cell lines such as MCF-7, MDA-MB-231 and 4T1 were evaluated by MTT assay. The result in MCF-7 cell line exhibited that transfection of BRCA 1/2 plasmids with CA NPs significantly reduced cancer cell growth in comparison to control group. Moreover, we noticed a likely pattern of cellular cytotoxicity in 4T1 murine cancer cell line. Following transfection with BRCA1 plasmid-loaded NPs, and Western blot analysis, a notable reduction in the phospho-MAPK protein of MAPK signaling pathway was detected, revealing reduced growth signal. Furthermore, in vivo study in 4T1 induced breast cancer mouse model showed that the tumor growth rate and final volume were decreased significantly in the mouse group treated intravenously with BRCA1 + NPs and BRCA2 + NPs formulations. Our results established that BRCA1/2 plasmids incorporated into CA NPs mitigated breast tumor growth, signifying their application in the therapy for breast cancer.

PEGylated Strontium Sulfite Nanoparticles with Spontaneously Formed Surface-Embedded Protein Corona Restrict Off-Target Distribution and Accelerate Breast Tumour-Selective Delivery of siRNA.

As transporters of RNAi therapeutics in preclinical and clinical studies, the application of nanoparticles is often hindered by their susceptibility to opsonin-mediated clearance, poor biological stability, ineffectual targeting, and undesirable effects on healthy cells. Prolonging the blood circulation time while minimizing the off-target distribution and associated toxicity is indispensable for the establishment of a clinically viable delivery system for therapeutic small interfering RNAs (siRNAs). Herein, we report a scalable and straightforward approach to fabricate non-toxic and biodegradable pH-responsive strontium sulfite nanoparticles (SSNs) wrapped with a hydrophilic coating material, biotinylated PEG to lessen unforeseen biological interactions. Surface functionalization of SSNs with PEG led to the generation of small and uniformly distributed particles with a significant affinity towards siRNAs and augmented internalization into breast cancer cells. A triple quadrupole liquid chromatography-mass spectrometry (LC-MS) was deployed to identify the proteins entrapped onto the SSNs, with the help of SwissProt.Mus_musculus database. The results demonstrated the reduction of opsonin proteins adsorption owing to the stealth effect of PEG. The distribution of PEGylated SSNs in mice after 4 h and 24 h of intravenous administration in breast tumour-bearing mice was found to be significantly less to the organs of the reticuloendothelial system (RES) and augmented accumulation in the tumour region. The anti-EGFR siRNA-loaded PEG-SSNs exerted a significant inhibitory effect on tumour development in the murine breast cancer model without any significant toxicity to healthy tissues. Therefore, PEGylated SSNs open up a new avenue for tumour-selective efficient delivery of siRNAs in managing breast cancer.

Scope and challenges of nanoparticle-based mRNA delivery in cancer treatment.

Messenger RNA (mRNA) recently emerged as an appealing alternative to treat and prevent diseases ranging from cancer and Alzheimer's disease to COVID-19 with significant clinical outputs. The in vitro-transcribed mRNA has been engineered to mimic the structure of natural mRNA for vaccination, cancer immunotherapy and protein replacement therapy. In past decades, significant progress has been noticed in unveiling the molecular pathways of mRNA, controlling its translatability and stability, and its evolutionary defense mechanism. However, numerous unsolved structural, biological, and technical difficulties hamper the successful implementation of systemic delivery of mRNA for safer human consumption. Advances in designing and manufacturing mRNA and selecting innovative delivery vehicles are mandatory to address the unresolved issues and achieve the full potential of mRNA drugs. Despite the substantial efforts made to improve the intracellular delivery of mRNA drugs, challenges associated with diverse applications in different routes still exist. This study examines the current progress of mRNA therapeutics and advancements in designing biomaterials and delivery strategies, the existing translational challenges of clinical tractability and the prospects of overcoming any challenges related to mRNA.

Evolving therapeutic proteins to precisely kill cancer cells.

The established cancer treatment strategy in clinical setting is based on chemo and radiation therapy, having limitations due to severe side-effects and drug-resistance. Small molecule chemo-drugs target any fast-dividing cells irrespective of healthy or defective origin. As a result, a substantial amount of healthy tissue is also destroyed. Moreover, failure to recognize the heterogeneity of tumour tissue results in drug-resistance over the course of time. On the other hand, peptides and proteins actively target somatic changes that are signature to any specific tumour tissue. Development and metastasis of cancer cells require unique disruption/alteration of protein activity. Identification of those wild and cancerous genotypes and phenotypes is the key to establishing easy 'targets' for protein based targeted therapeutics. The approach is cytostatic and tissue specific, which reduces drug toxicity. Biopharmaceutical products based on proteins and peptides are slowly re-directing oncology from cytotoxic small molecular treatment approach to target oriented cytostatic strategy. This review focuses on current and upcoming peptide and protein-based precision therapeutics. At the same time, the study also shades light on the technological advancement in the field of protein and peptide-based therapeutics.

Natural and Synthetic Micelles for the Delivery of Small Molecule Drugs, Imaging Agents and Nucleic Acids.

The poor solubility, lack of targetability, quick renal clearance, and degradability of many therapeutic and imaging agents strongly limit their applications inside the human body. Amphiphilic copolymers having self-assembling properties can form core-shell structures called micelles, a promising nanocarrier for hydrophobic drugs, plasmid DNA, oligonucleotides, small interfering RNAs (siRNAs), and imaging agents. Fabrication of micelles loaded with different pharmaceutical agents provides numerous advantages, including therapeutic efficacy, diagnostic sensitivity, and controlled release to the desired tissues. Moreover, their smaller particle size (10-100 nm) and modified surfaces with different functional groups (such as ligands) help them to accumulate easily in the target location, enhancing cellular uptake and reducing unwanted side effects. Furthermore, the release of the encapsulated agents may also be triggered from stimuli-sensitive micelles under different physiological conditions or by an external stimulus. In this review article, we discuss the recent advancements in formulating and targeting of different natural and synthetic micelles, including block copolymer micelles, cationic micelles, and dendrimers-, polysaccharide- and protein-based micelles for the delivery of different therapeutic and diagnostic agents. Finally, their applications, outcomes, and future perspectives have been summarized.

Clinical Outcomes of Chemotherapeutic Molecules as Single and Multiple Agents in Advanced Non-Small-Cell Lung Carcinoma (NSCLC) Patients.

Background and Objectives: Lung cancer is the second most common cancer in the world. Non-small-cell lung carcinoma (NSCLC) makes up 85% of all lung cancer cases and the majority of patients are diagnosed when the cancer is advanced. Over the years, many anticancer drugs have been designed and introduced into the market to treat patients with advanced NSCLC. This review aims to discuss the comparative therapeutic benefits of conventional chemotherapeutics and other drugs available for treating advanced NSCLC. Materials and Methods: A literature search for first-line treatment of advanced NSCLC was carried out on PubMed and Google Scholar. Objective response rate (ORR) and overall survival were chosen as target endpoints. Results: Monotherapy showed lower treatment endpoints compared to combination therapy. Different combinations of platinum-based doublets demonstrated similar efficacies in treating NSCLC. However, pemetrexed-platinum doublets showed significantly better treatment endpoint in patients with non-squamous NSCLC. Most studies showing the best complete response rate (CRR) utilized epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI), while most studies producing the best overall survival included programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) inhibitors in their treatment regimens. Conclusions: The findings of this review indicate that targeted therapy using specific inhibitors is now the most promising first-line anticancer treatment available in the market. However, chemotherapy is still effective in treating advanced NSCLC and is viable as a first-line treatment.

PH-responsive strontium nanoparticles for targeted gene therapy against mammary carcinoma cells.

Genetic intervention via the delivery of functional genes such as plasmid DNA (pDNA) and short-interfering RNA (siRNA) offers a great way to treat many single or multiple genetic defects effectively, including mammary carcinoma. Delivery of naked therapeutic genes or siRNAs is, however, short-lived due to biological clearance by scavenging nucleases and circulating monocytes. Low cellular internalization of negatively-charged nucleic acids further causes low transfection or silencing activity. Development of safe and effectual gene vectors is therefore undeniably crucial to the success of nucleic acid delivery. Inorganic nanoparticles have attracted considerable attention in the recent years due to their high loading capacity and encapsulation activity. Here we introduce strontium salt-based nanoparticles, namely, strontium sulfate, strontium sulfite and strontium fluoride as new inorganic nanocarriers. Generated strontium salt particles were found to be nanosized with high affinity towards negatively-charged pDNA and siRNA. Degradation of the particles was seen with a drop in pH, suggesting their capacity to respond to pH change and undergo dissolution at endosomal pH to release the genetic materials. While the particles are relatively nontoxic towards the cells, siRNA-loaded SrF2 and SrSO3 particles exerted superior transgene expression and knockdown activity of MAPK and AKT, leading to inhibition of their phosphorylation to a distinctive extent in both MCF-7 and 4T1 cells. Strontium salt nanoparticles have thus emerged as a promising tool for applications in cancer gene therapy.

Nanoparticles Targeting Receptors on Breast Cancer for Efficient Delivery of Chemotherapeutics.

The journey of chemotherapeutic drugs from the site of administration to the site of action is confronted by several factors including low bioavailability, uneven distribution in major organs, limited accessibility of drug molecules to the distant tumor tissues, and lower therapeutic indexes. These unavoidable features of classical chemotherapeutics necessitate an additional high, repetitive dose of drugs to obtain maximum therapeutic responses with the result of unintended adverse side effects. An erratic tumor microenvironment, notable drawbacks of conventional chemotherapy, and multidrug-resistant mechanisms of breast cancer cells warrant precisely designed therapeutics for the treatment of cancers. In recent decades, nanoparticles have been deployed for the delivery of standard anticancer drugs to maximize the therapeutic potency while minimizing the adverse effects to increase the quality and span of life. Several organic and inorganic nanoplatforms that have been designed exploiting the distinctive features of the tumor microenvironment and tumor cells offer favorable physicochemical properties and pharmacokinetic profiles of a parent drug, with delivery of higher amounts of the drug to the pathological site and its controlled release, thereby improving the balance between its efficacy and toxicity. Advances to this front have included design and construction of targeted nanoparticles by conjugating homing devices like peptide, ligand, and Fab on the surface of nanomaterials to navigate nanoparticledrug complexes towards the target tumor cell with minimal destruction of healthy cells. Furthermore, actively targeting nanoparticles can facilitate the delivery and cellular uptake of nanoparticle-loaded drug constructs via binding with specific receptors expressed aberrantly on the surface of a tumor cell. Herein, we present an overview of the principle of targeted delivery approaches, exploiting drug-nanoparticle conjugates with multiple targeting moieties to target specific receptors of breast cancer cells and highlighting therapeutic evaluation in preclinical studies. We conclude that an understanding of the translational gap and challenges would show the possible future directions to foster the development of novel targeted nanotherapeutics.

Molecular endoscopic imaging for the detection of Barrett's metaplasia using biodegradable inorganic nanoparticles: An ex-vivo pilot study on human tissue.

BACKGROUND AND STUDY AIMS: Despite major technical advancements, endoscopic surveillance for detecting premalignant lesions in Barrett's esophagus is challenging because of their flat appearance with only subtle morphological changes. Molecular endoscopic imaging (MEI) using nanoparticles (NPs), coupled with fluorescently labeled antibody permits visualization of disease-specific molecular alterations. The aim of this ex vivo study was to assess the diagnostic applicability of MEI with NPs to detect Barrett's metaplasia. PATIENTS AND METHODS: Seven patients undergoing endoscopic surveillance of known Barrett's esophagus were recruited. Freshly resected biopsy specimens were incubated with NPs coupled with FITC labeled Muc-2 antibodies and examined with MEI. Fluorescence intensity from Barrett's mucosa and control specimens were compared, followed by histological confirmation. RESULTS: Fluorescence signals, indicating the presence of goblet cells, were noted for traditional MEI using Muc-2 antibodies in Barrett's intestinal metaplasia. Significantly stronger fluorescence signals were achieved with NPs coupled with FITC-conjugated Muc-2 antibodies. The results of MEI with NPs for the prediction of Barrett's metaplasia correlated with the final histopathological examination in all the cases. CONCLUSIONS: Highly-specific NPs detected Barrett's metaplasia more efficiently than conventional MEI in this first feasibility study. MEI was as effective as standard histopathology for identifying Muc-2 containing goblet cells for diagnosis of Barrett's metaplasia. (DRKS-ID: DRKS00017747).

Optimization and Formulation of Nanostructured and Self-Assembled Caseinate Micelles for Enhanced Cytotoxic Effects of Paclitaxel on Breast Cancer Cells.

BACKGROUND: Paclitaxel (PTX) is a widely used anti-cancer drug for treating various types of solid malignant tumors including breast, ovarian and lung cancers. However, PTX has a low therapeutic response and is linked with acquired resistance, as well as a high incidence of adverse events, such as allergic reactions, neurotoxicity and myelosuppression. The situation is compounded when its complex chemical structure contributes towards hydrophobicity, shortening its circulation time in blood, causing off-target effects and limiting its therapeutic activity against cancer cells. Formulating a smart nano-carrier may overcome the solubility and toxicity issues of the drug and enable its more selective delivery to the cancerous cells. Among the nano-carriers, natural polymers are of great importance due to their excellent biodegradability, non-toxicity and good accessibility. The aim of the present research is to develop self-assembled sodium caseinate nanomicelles (NaCNs) with PTX loaded into the hydrophobic core of NaCNs for effective uptake of the drug in cancer cells and its subsequent intracellular release. METHODS: The PTX-loaded micelle was characterized with high-performance liquid chromatography (HPLC), Fourier Transform Infrared Spectra (FTIR), High Resolution-Transmission Electron Microscope (HR-TEM), Field Emission Scanning Electron Microscope (FESEM) and Energy Dispersive X-Ray (EDX). Following treatment with PTX-loaded NaCNs, cell viability, cellular uptake and morphological changes were analyzed using MCF-7 and MDA-MB 231 human breast cancer cell lines. RESULTS: We found that PTX-loaded NaCNs efficiently released PTX in an acidic tumor environment, while showing an enhanced cytotoxicity, cellular uptake and in-vivo anti-tumor efficacy in a mouse model of breast cancer when compared to free drug and blank micelles. Additionally, the nanomicelles also presented improved colloidal stability for three months at 4 °C and -20 °C and when placed at a temperature of 37 °C. CONCLUSIONS: We conclude that the newly developed NaCNs is a promising carrier of PTX to enhance tumor accumulation of the drug while addressing its toxicity issues as well.

Characterization and Evaluation of Bone-Derived Nanoparticles as a Novel pH-Responsive Carrier for Delivery of Doxorubicin into Breast Cancer Cells.

Background: The limitations of conventional treatment modalities in cancer, especially in breast cancer, facilitated the necessity for developing a safer drug delivery system (DDS). Inorganic nano-carriers based on calcium phosphates such as hydroxyapatite (HA) and carbonate apatite (CA) have gained attention due to their biocompatibility, reduced toxicity, and improved therapeutic efficacy. Methods: In this study, the potential of goose bone ash (GBA), a natural derivative of HA or CA, was exploited as a pH-responsive carrier to successfully deliver doxorubicin (DOX), an anthracycline drug into breast cancer cells (e.g., MCF-7 and MDA-MB-231 cells). GBA in either pristine form or in suspension was characterized in terms of size, morphology, functional groups, cellular internalization, cytotoxicity, pH-responsive drug (DOX) release, and protein corona analysis. Results: The pH-responsive drug release study demonstrated the prompt release of DOX from GBA through its disintegration in acidic pH (5.5-6.5), which mimics the pH of the endosomal and lysosomal compartments as well as the stability of GBA in physiological pH (pH 7.5). The result of DOX binding with GBA indicated an increment in binding affinity with increasing concentrations of DOX. Cell viability and cytotoxicity analysis showed no innate toxicity of GBA particles. Both qualitative and quantitative cellular uptake analysis in both cell lines displayed an enhanced cellular internalization of DOX-loaded GBA compared to free DOX molecules. The protein corona spontaneously formed on the surface of GBA particles exhibited its affinity toward transport proteins, structural proteins, and a few other selective proteins. The adsorption of transport proteins could extend the circulation half-life in biological environment and increase the accumulation of the drug-loaded NPs through the enhanced permeability and retention (EPR) effect at the tumor site. Conclusion: These findings highlight the potential of GBA as a DDS to successfully deliver therapeutics into breast cancer cells.

Carbonate Apatite and Hydroxyapatite Formulated with Minimal Ingredients to Deliver SiRNA into Breast Cancer Cells In Vitro and In Vivo.

INTRODUCTION: Cancer is one of the top-ranked noncommunicable diseases causing deaths to nine million people and affecting almost double worldwide in 2018. Tremendous advancement in surgery, chemotherapy, radiation and targeted immunotherapy have improved the rate of cure and disease-free survival. As genetic mutations vary in different cancers, potential of customized treatment to silence the problem gene/s at the translational level is being explored too. Yet delivering therapeutics at the required dosage only to the affected cells without affecting the healthy ones, is a big hurdle to be overcome. Scientists worldwide have been working to invent a smart drug delivery system for targeted delivery of therapeutics to tumor tissues only. As part of such an effort, few organic nanocarriers went to clinical trials, while inorganic nanoparticles (NPs) are still in development stage despite their many customizable properties. Carbonate apatite (CA), a pH sensitive nanocarrier has emerged as an efficient delivery system for drugs, plasmids and siRNAs in preclinical models of breast and colon cancers. Like hydroxyapatite (HA) which serves as a classical tool for delivery of genetic materials such as siRNA and plasmid, CA is an apatite-based synthetic carrier. We developed simplified methods of formulating CA-in-DMEM and a DMEM-mimicking buffer and HA in a HEPES-buffered solution and characterized them in terms of size, stability, protein corona (PC) composition, cytotoxicity, siRNA delivery efficiency in breast cancer cells and siRNA biodistribution profile in a mouse model of breast cancer. METHODS: Particle growth was analyzed via spectrophotometry and light microscopy, size was measured via dynamic light scattering and scanning electron microscopy and confirmation of functional groups in apatite structures was made by FT-IR. siRNA-binding was analyzed via spectrophotometry. Stability of the formulation solutions/buffers was tested over various time points and at different temperatures to determine their compatibility in the context of practical usage. Cellular uptake was studied via fluorescence microscopy. MTT assay was performed to measure the cytotoxicity of the NPs. Liquid chromatography-mass spectrometry was carried out to analyze the PC formed around all three different NPs in serum-containing media. To explore biodistribution of all the formulations, fluorescence-labeled siRNA-loaded NPs were administered intravenously prior to analysis of fluorescence intensity in the collected organs and tumors of the treated mice. RESULTS: The size of NPs in 10% serum-containing media was dramatically different where CA-in-DMB and HA were much larger than CA-in-DMEM. Effect of media was notable on the PC composition of all three NPs. All three NPs bound albumin and some common protease inhibitors involved in bone metabolism due to their compositional similarity to our bone materials. Moreover, CA also bound heme-binding proteins and opsonins. Unlike CA, HA bound different kinds of keratins. Difference in PC constitution was likely to influence accumulation of NPs in various organs including those of reticuloendothelial system, such as liver and spleen and the tumor. We found 10 times more tumor accumulation of CA-in-DMB than CA-in-DMEM, which could be due to more stable siRNA-binding and distinct PC composition of the former. CONCLUSION: As a nanocarrier CA is more efficient than HA for siRNA delivery to the tumor. CA prepared in a buffer containing only the mere constituents was potentially more efficient than classical CA prepared in DMEM, owing to the exclusion of interference attributed by the inorganic ions and organic molecules present in DMEM.

Insulin-Loaded Barium Salt Particles Facilitate Oral Delivery of Insulin in Diabetic Rats.

Oral delivery is considered as the most preferred and yet most challenging mode of drug administration; especially a fragile and sensitive peptide like insulin that shows extremely low bioavailability through the gastro-intestinal (GIT) route. To address this problem, we have designed a novel drug delivery system (DDS) using precipitation-induced Barium (Ba) salt particles. The DDS can load insulin molecules and transport them through the GIT route. There were several in vitro simulation tests carried out to prove the efficiency of Ba salt particles as oral delivery candidates. All three Ba salt particles (BaSO4, BaSO3, and BaCO3) showed very good loading of insulin (>70% in all formulations) and a degree of resistance throughout a wide range of pHs from basic to acidic conditions when assessed by spectrophotometry. Particles and insulin-associated particles were morphologically assessed and characterized using FE-SEM and FT-IR. A set of tests were designed and carried out with mucin to predict whether the particles are potentially capable of overcoming one of the barriers for crossing intestinal epithelium. The mucin binding experiment demonstrated 60-100% of mucin adhesion to the three different particles. FT-IR identifies the characteristic peaks for mucin protein, particles, and particle-mucin complex re-confirming mucin adhesion to the particles. Finally, the effectiveness of nano-insulin was tested on streptozotocin (STZ) induced diabetic rats. A short acting human insulin analog, insulin aspart, was loaded into Ba salt particles at a dose of 100 IU/Kg prior to oral administration. Among the three formulations, insulin aspart-loaded BaSO4 and BaCO3 particles dramatically reduced the existing hyperglycemia. BaSO4 with loaded Insulin showed an onset of glucose-lowering action within 1 hr, with blood glucose level measured significantly lower compared to the 2nd and 3rd h (p < 0.05). Insulin-loaded BaCO3 particles showed a significant decrease in blood glucose level at 1-2 h, although the glucose level started to show a slight rise at 3rd h and by 4th h, it was back to baseline level. However, although BaSO3 particles with loaded insulin showed a trend of reduction in blood glucose level, the reduction was not found to be significant (p < 0.05) at any point in time. Therefore, oral formulations of insulin/BaSO4 and insulin/BaCO3 particles were observed as effective as native insulin aspart subcutaneous formulation in terms of onset and duration of action. Further investigation will be needed to reveal bioavailability and mechanism of action of this novel Nano-Insulin formulations.

Fe/Mg-Modified Carbonate Apatite with Uniform Particle Size and Unique Transport Protein-Related Protein Corona Efficiently Delivers Doxorubicin into Breast Cancer Cells.

Breast cancer is the abnormal, uncontrollable proliferation of cells in the breast. Conventional treatment modalities like chemotherapy induce deteriorating side effects on healthy cells. Non-viral inorganic nanoparticles (NPs) confer exclusive characteristics, such as, stability, controllable shape and size, facile surface modification, and unique magnetic and optical properties which make them attractive drug carriers. Among them, carbonate apatite (CA) particles are pH-responsive in nature, enabling rapid intracellular drug release, but are typically heterogeneous with the tendency to self-aggregate. Here, we modified the nano-carrier by partially substituting Ca2+ with Mg2+ and Fe3+ into a basic lattice structure of CA, forming Fe/Mg-carbonate apatite (Fe/Mg-CA) NPs with the ability to mitigate self-aggregation, form unique protein corona in the presence of serum and efficiently deliver doxorubicin (DOX), an anti-cancer drug into breast cancer cells. Two formulations of Fe/Mg-CA NPs were generated by adding different concentrations of Fe3+ and Mg2+ along with a fixed amount of Ca2+ in bicarbonate buffered DMEM (Dulbecco's Modified Eagle's Medium), followed by 30 min incubation at 37 °C. Particles were characterized by turbidity analysis, z-average diameter and zeta potential measurement, optical microscopy, field emission scanning electron microscopy (FESEM), Fourier transform infrared spectroscopy (FTIR), energy dispersive X-ray (EDX), flame atomic absorption spectroscopy (FAAS), pH dissolution, drug binding, cellular uptake, thiazolyl blue tetrazolium bromide (MTT) assay, stability analysis, and protein corona study by LCMS (Liquid chromatography-mass spectrometry). Both formulations of Fe/Mg-CA displayed mostly uniform nano-sized particles with less tendency to aggregate. The EDX and FAAS elemental analysis confirmed the weight (%) of Ca, Fe and Mg, along with their Ca/P ratio in the particles. A constant drug binding efficiency was noticed with 5 µM to 10 µM of initial DOX concentration. A pH dissolution study of Fe/Mg-CA NPs revealed the quick release of DOX in acidic pH. Enhancement of cytotoxicity for the chemotherapy drug was greater for Fe/Mg-CA NPs as compared to CA NPs, which could be explained by an increase in cellular internalization as a result of the small z-average diameter of the former. The protein corona study by LCMS demonstrated that Fe/Mg-CA NPs exhibited the highest affinity towards transport proteins without binding with opsonins. Biodistribution study was performed to study the effect of DOX-loaded Fe/Mg-CA NPs on the tissue distribution of DOX in Balb/c 4T1 tumor-bearing mice. Both formulations of Fe/Mg-CA NPs have significantly increased the accumulation of DOX in tumors. Interestingly, high Fe/Mg-CA NPs exhibited less off-target distribution compared to low Fe/Mg-CA NPs. Furthermore, the blood plasma analysis revealed prolonged blood circulation half-life of DOX-loaded low and high Fe/Mg-CA NPs compared to free DOX solution. Modifying CA NPs with Fe3+ and Mg2+, thereby, led to the generation of nano-sized particles with less tendency to aggregate, enhancing the drug binding efficiency, cellular uptake, and cytotoxicity without hampering drug release in acidic pH, while improving the circulation half-life and tumor accumulation of DOX. Therefore, Fe/Mg-CA which predominantly forms a transport protein-related protein corona could be a proficient carrier for therapeutic delivery in breast cancer.

Krebs Cycle Intermediate-Modified Carbonate Apatite Nanoparticles Drastically Reduce Mouse Tumor Burden and Toxicity by Restricting Broad Tissue Distribution of Anticancer Drugs.

The morphology, size, and surface area of nanoparticles (NPs), with the existence of functional groups on their surface, contribute to the drug binding affinity, distribution of the payload in different organs, and targeting of a particular tumor for exerting effective antitumor activity in vivo. However, the inherent chemical structure of NPs causing unpredictable biodistribution with a toxic outcome still poses a serious challenge in clinical chemotherapy. In this study, carbonate apatite (CA), citrate-modified CA (CMCA) NPs, and α-ketoglutaric acid-modified CA (α-KAMCA) NPs were employed as carriers of anticancer drugs for antitumor, pharmacokinetic, and toxicological analysis in a murine breast cancer model. The results demonstrated almost five-fold enhanced tumor regression in the cyclophosphamide (CYP)-loaded α-KAMCA NP-treated group compared to the group treated with CYP only. Also, NPs promoted much higher drug accumulation in blood and tumor in comparison with the drug injected without a carrier. In addition, doxorubicin (DOX)-loaded NPs exhibited less accumulation in the heart, indicating less potential myocardial toxicity in mice compared to free DOX. Our findings, thus, conclude that CA, CMCA, and α-KAMCA NPs extended the circulation half-life and enhanced the anticancer effect with reduced toxicity of conventional chemotherapeutics in healthy organs, signifying that they are promising drug delivery devices in breast cancer treatment.

Intracellular Delivery of siRNAs Targeting AKT and ERBB2 Genes Enhances Chemosensitization of Breast Cancer Cells in a Culture and Animal Model.

: Pharmacotherapy as the mainstay in the management of breast cancer suffers from various drawbacks, including non-targeted biodistribution, narrow therapeutic and safety windows, and also resistance to treatment. Thus, alleviation of the constraints from the pharmacodynamic and pharmacokinetic profile of classical anti-cancer drugs could lead to improvements in efficacy and patient survival in malignancies. Moreover, modifications in the genetic pathophysiology of cancer via administration of small nucleic acids might pave the way towards higher response rates to chemotherapeutics. Inorganic pH-dependent carbonate apatite (CA) nanoparticles were utilized in this study to efficiently deliver various classes of therapeutics into cancer cells. Co-delivery of drugs and genetic materials was successfully attained through a carbonate apatite delivery device. On 4T1 cells, siRNAs against AKT and ERBB2 plus paclitaxel or docetaxel resulted in the largest increase in anti-cancer effects compared to CA/paclitaxel or CA/docetaxel. Therefore, these ingredients were selected for further in vivo investigations. Animals receiving injections of CA/paclitaxel or CA/docetaxel loaded with siRNAs against AKT and ERBB2 possessed significantly smaller tumors compared to CA/drug-treated mice. Interestingly, synergistic interactions in target protein knock down with combinations of CA/AKT/paclitaxel, CA/ERBB2/docetaxel were documented via western blotting.

Targeting Cell Adhesion Molecules via Carbonate Apatite-Mediated Delivery of Specific siRNAs to Breast Cancer Cells In Vitro and In Vivo.

While several treatment strategies are applied to cure breast cancer, it still remains one of the leading causes of female deaths worldwide. Since chemotherapeutic drugs have severe side effects and are responsible for development of drug resistance in cancer cells, gene therapy is now considered as one of the promising options to address the current treatment limitations. Identification of the over-expressed genes accounting for constitutive activation of certain pathways, and their subsequent knockdown with specific small interfering RNAs (siRNAs), could be a powerful tool in inhibiting proliferation and survival of cancer cells. In this study, we delivered siRNAs against mRNA transcripts of over-regulated cell adhesion molecules such as catenin alpha 1 (CTNNA1), catenin beta 1 (CTNNB1), talin-1 (TLN1), vinculin (VCL), paxillin (PXN), and actinin-1 (ACTN1) in human (MCF-7 and MDA-MB-231) and murine (4T1) cell lines as well as in the murine female Balb/c mice model. In order to overcome the barriers of cell permeability and nuclease-mediated degradation, the pH-sensitive carbonate apatite (CA) nanocarrier was used as a delivery vehicle. While targeting CTNNA1, CTNNB1, TLN1, VCL, PXN, and ACTN1 resulted in a reduction of cell viability in MCF-7 and MDA-MB-231 cells, delivery of all these siRNAs via carbonate apatite (CA) nanoparticles successfully reduced the cell viability in 4T1 cells. In 4T1 cells, delivery of CTNNA1, CTNNB1, TLN1, VCL, PXN, and ACTN1 siRNAs with CA caused significant reduction in phosphorylated and total AKT levels. Furthermore, reduced band intensity was observed for phosphorylated and total MAPK upon transfection of 4T1 cells with CTNNA1, CTNNB1, and VCL siRNAs. Intravenous delivery of CTNNA1 siRNA with CA nanoparticles significantly reduced tumor volume in the initial phase of the study, while siRNAs targeting CTNNB1, TLN1, VCL, PXN, and ACTN1 genes significantly decreased the tumor burden at all time points. The tumor weights at the end of the treatments were also notably smaller compared to CA. This successfully demonstrates that targeting these dysregulated genes via RNAi and by using a suitable delivery vehicle such as CA could serve as a promising therapeutic treatment modality for breast cancers.

Oncogenic Signaling in Tumorigenesis and Applications of siRNA Nanotherapeutics in Breast Cancer.

Overexpression of oncogenes and cross-talks of the oncoproteins-regulated signaling cascades with other intracellular pathways in breast cancer could lead to massive abnormal signaling with the consequence of tumorigenesis. The ability to identify the genes having vital roles in cancer development would give a promising therapeutics strategy in combating the disease. Genetic manipulations through siRNAs targeting the complementary sequence of the oncogenic mRNA in breast cancer is one of the promising approaches that can be harnessed to develop more efficient treatments for breast cancer. In this review, we highlighted the effects of major signaling pathways stimulated by oncogene products on breast tumorigenesis and discussed the potential therapeutic strategies for targeted delivery of siRNAs with nanoparticles in suppressing the stimulated signaling pathways.

Transfection with p21 and p53 tumor suppressor plasmids suppressed breast tumor growth in syngeneic mouse model.

Treatment of breast cancer by delivering important tumor suppressor plasmids is a promising approach in the field of clinical medicine. We transfected p21 and p53 tumor suppressor plasmids, into different breast cancer cell lines using inorganic nanoparticles (NPs) of carbonate apatite to evaluate the effect of gene expression on reducing breast cancer cell growth. In triple negative MDA-MB-231 breast cancer cell line, the cytotoxicity assay upon combined delivery of p21 and p53 plasmid loaded NPs showed significant decrease in cell growth compared to distinct p21 or p53 treatments. Also, in MCF-7 and 4T1 cell lines, significant reduction in cellular growth was observed following p21 or p53 plasmid transfection. The Western blot data showed that NP loaded p21 and p53 transgene delivery in MDA-MB-231 cell line resulted in a noteworthy decrease in phosphorylated form of MAPK protein of MAPK/ERK pathway. The in vivo studies in syngeneic breast cancer mouse model demonstrated that the rate of growth and final tumor volume were reduced to a greater extent in mice that received intravenous injection of p21 + NP and p53 + NP therapeutics.