Synergistic Antitumor Potency of a Self-Assembling Cyclodextrin Nanoplex for the Co-Delivery of 5-Fluorouracil and Interleukin-2 in the Treatment of Colorectal Cancer.

Chemotherapy is the most used method after surgery in the treatment of colon cancer. Cancer cells escape the recognition mechanism of immune system cells to survive and develop chemoresistance. Therefore, the use of immunotherapy in combination with chemotherapy can increase the effectiveness of the treatment. Nanoparticles have been used clinically to increase the accumulation of therapeutics in target tissues and reduce toxicity. In this paper, nanoplexes were formed via cationic cyclodextrin polymer, 5-Fluorouracil, and Interleukin-2 based on the opposite charge interaction of macromolecules without undergoing any structural changes or losing the biological activity of Interleukin-2. Anticancer activities of nanoplexes were determined in two-dimensional and three-dimensional cell culture setups. The dual drug-loaded cyclodextrin nanoplexes diffused deeper into the spheroids and accelerated apoptosis when compared with 5-FU solutions. In the colorectal tumor-bearing animal model, survival rate, antitumor activity, metastasis, and immune response parameters were assessed using a cyclodextrin derivative, which was found to be safe based on the ALT/AST levels in healthy mice. Histomorphometric analysis showed that the groups treated with the nanoplex formulation had significantly fewer initial tumors and lung foci when compared with the control. The dual drug-loaded nanoplex could be a promising drug delivery technique in the immunochemotherapy of colorectal cancer.

Insight into oral amphiphilic cyclodextrin nanoparticles for colorectal cancer: comprehensive mathematical model of drug release kinetic studies and antitumoral efficacy in 3D spheroid colon tumors.

Colorectal cancer (CRC) is the third most diagnosed cancer type globally and ranks second in cancer-related deaths. With the current treatment possibilities, a definitive, safe, and effective treatment approach for CRC has not been presented yet. However, new drug delivery systems show promise in this field. Amphiphilic cyclodextrin-based nanocarriers are innovative and interesting formulation approaches for targeting the colon through oral administration. In our previous studies, oral chemotherapy for colon tumors was aimed and promising results were obtained with formulation development studies, mucin interaction, mucus penetration, cytotoxicity, and permeability in 2D cell culture, and furthermore in vivo antitumoral and antimetastatic efficacy in early and late-stage colon cancer models and biodistribution after single dose oral administration. This study was carried out to further elucidate oral camptothecin (CPT)-loaded amphiphilic cyclodextrin nanoparticles for the local treatment of colorectal tumors in terms of their drug release behavior and efficacy in 3-dimensional tumor models to predict the in vivo efficacy of different nanocarriers. The main objective was to build a bridge between formulation development and in vitro phase and animal studies. In this context, CPT-loaded polycationic-ß-cyclodextrin nanoparticles caused reduced cell viability in CT26 and HT29 colon carcinoma spheroid tumors of mice and human origin, respectively. In addition, the release profile, which is one of the critical quality parameters in new drug delivery systems, was investigated mathematically by release kinetic modeling for the first time. The overall findings indicated that the strategy of orally targeting anticancer drugs such as CPT with positively charged poly-ß-CD-C6 nanoparticles to colon tumors for local and/or systemic efficacy is a promising approach.

A different approach to immunochemotherapy for colon Cancer: Development of nanoplexes of cyclodextrins and Interleukin-2 loaded with 5-FU.

Immune system deficiencies are crucial in the progression of cancer, predominantly because immune cells are not stimulated by cytokines to eradicate cancer cells. Immunochemotherapy is currently considered an innovative approach that creates pathways in cancer treatment, sometimes also aiding in the efficacy of chemotherapeutics. The aim of this study was to prepare a cyclodextrin (CD) nanoplex based on charge interaction to deliver the anticancer drug 5-fluorouracil (5-FU) and Interleukin-2 (IL-2), thereby forming a nanoscale drug delivery system aimed at chemo-immunotherapy for colorectal cancers. The CD:IL-2 nanoplexes were obtained with a particle size below 100 nm and a cationic surface charge based on the extent of charge interaction of the cationic CD polymer with negatively charged IL-2. The loading capacity of CD nanoplexes was 40% for 5-FU and 99.8% for IL-2. Nanoplexes maintained physical stability in terms of particle size and zeta potential in aqueous solution for 1 week at + 4 °C. Moreover, the structural integrity of IL-2 loaded into CD nanoplexes was confirmed by SDS-PAGE analysis. The cumulative release rates of both 5-FU and IL-2 were found to be more than 80% in simulated biological fluids in 12 h. Cell culture studies demonstrate that CD polymers are safe on healthy L929 mouse fibroblast cells. Drug-loaded CD nanoplexes were determined to have a higher anticancer effect than free drug solution against CT26 mouse colon carcinoma cells. In addition, intestinal permeability studies supported the conclusion that CD nanoplexes could be promising candidates for oral chemotherapy as well. In conclusion, effective cancer therapy utilizing the absorptive/cellular uptake effect of CDs, the synergic effect and co-transport of chemotherapeutic drugs and immunotherapeutic molecules is a promising approach. Furthermore, the transport of IL-2 with this nano-sized system can reduce or avoid its toxicity problem in the clinic.

A Review on Cancer Immunotherapy and Applications of Nanotechnology to Chemoimmunotherapy of Different Cancers.

Clinically, different approaches are adopted worldwide for the treatment of cancer, which still ranks second among all causes of death. Immunotherapy for cancer treatment has been the focus of attention in recent years, aiming for an eventual antitumoral effect through the immune system response to cancer cells both prophylactically and therapeutically. The application of nanoparticulate delivery systems for cancer immunotherapy, which is defined as the use of immune system features in cancer treatment, is currently the focus of research. Nanomedicines and nanoparticulate macromolecule delivery for cancer therapy is believed to facilitate selective cytotoxicity based on passive or active targeting to tumors resulting in improved therapeutic efficacy and reduced side effects. Today, with more than 55 different nanomedicines in the market, it is possible to provide more effective cancer diagnosis and treatment by using nanotechnology. Cancer immunotherapy uses the body's immune system to respond to cancer cells; however, this may lead to increased immune response and immunogenicity. Selectivity and targeting to cancer cells and tumors may lead the way to safer immunotherapy and nanotechnology-based delivery approaches that can help achieve the desired success in cancer treatment.

Erlotinib complexation with randomly methylated ß-cyclodextrin improves drug solubility, intestinal permeability, and therapeutic efficacy in non-small cell lung cancer.

The purpose of this study was to investigate the impact of anticancer drug erlotinib-randomly methylated-ß-cyclodextrin complex (ERL-RAMEB CD) on drug solubility and dissolution rate. Phase solubility study showed erlotinib displayed maximum solubility in RAMEB CD solution and the stability constant (Kc) was calculated to be 370 ± 16 M-1. The optimal formulation was obtained with ERL-RAMEB CD in a 1:1 molar ratio using the co-lyophilization technique. Differential scanning calorimetry (DSC) and Scanning electron microscopy (SEM) verified the inclusion of complex formation. In vitro dissolution study confirmed ERL-RAMEB CD as a favorable approach to increase drug dissolution with a 1.5-fold increase than free ERL at 1 h. An improved dissolution with ∼88.4% drug release at 1 h was observed, in comparison with Erlotinib with ∼58.7% release in 45 min. The in vitro cytotoxicity results indicated that the ERL-RAMEB CD inclusion complex reduced cell viability than free erlotinib. Caco-2 cell uptake that is indicative of drug intestinal permeability resulted in a 5-fold higher uptake of ERL-RAMEB CD inclusion complex than the ERL solution. Hence, ERL-RAMEB CD complexation displays a strong potential to increase dissolution and permeability of erlotinib leading eventually to enhanced oral bioavailability.

Therapeutic Efficacy and Biodistribution of Paclitaxel-Bound Amphiphilic Cyclodextrin Nanoparticles: Analyses in 3D Tumor Culture and Tumor-Bearing Animals In Vivo.

The uniqueness of paclitaxel's antimitotic action mechanism has fueled research toward its application in more effective and safer cancer treatments. However, the low water solubility, recrystallization, and side effects hinder the clinical success of classic paclitaxel chemotherapy. The aim of this study was to evaluate the in vivo efficacy and biodistribution of paclitaxel encapsulated in injectable amphiphilic cyclodextrin nanoparticles of different surface charges. It was found that paclitaxel-loaded amphiphilic cyclodextrin nanoparticles showed an antitumoral effect earlier than the drug solution. Moreover, the blank nanoparticles reduced the tumor growth with a similar trend to the paclitaxel solution. At 24 h, the nanoparticles had not accumulated in the heart and lungs according to the biodistribution assessed by in vivo imaging. Therefore, our results indicated that the amphiphilic cyclodextrin nanoparticles are potentially devoid of cardiac toxicity, which limits the clinical use and commercialization of certain polymeric nanoparticles. In conclusion, the amphiphilic cyclodextrin nanoparticles with different surface charge increased the efficiency of paclitaxel in vitro and in vivo. Cyclodextrin nanoparticles could be a good candidate vehicle for intravenous paclitaxel delivery.

Polycationic cyclodextrin nanoparticles induce apoptosis and affect antitumoral activity in HepG2 cell line: An evaluation at the molecular level.

Hepatocellular carcinoma (HCC) is a highly metastatic primary liver cancer generating molecular alterations that end up escaping the apoptotic machinery and conferring multidrug resistance. Targeted medicines with increased and selective cytotoxicity and minimal drug resistance are essential for the treatment of HCC. In this study, a self-assembled polycationic (PC) amphiphilic ß-cyclodextrin (ßCDC6) nanoparticle formulation was characterized and its efficacy over HCC cell line HepG2 was evaluated in terms of cytotoxicity, apoptotic potential, chemosensitivity and mitochondrial balance utilizing biochemical, gene expression and proteomic approaches without encapsulating an anti-neoplastic agent. Blank PC ßCDC6 exerted an anti-proliferative effect on 3D multicellular HepG2 spheroid tumors. These nanoparticles were able to trigger apoptosis proved by caspase 3/7 activity, gene expression and flow cytometry studies. The subjection of PC restored the chemosensitivity of HepG2 cells by suppressing the function of p-glycoprotein. The proteomic studies with Q-TOF LC/MS revealed 73 proteins that are aberrantly encoded after cells were treated with the blank PC. Metabolomic analysis further confirmed the shift in certain biological pathways. Thus, we confirmed that the hepatocellular carcinoma-targeting ßCDC6 PC nanoparticles induce apoptosis, lower the rate of cell proliferation, hinder multidrug resistance and they are convenient carriers for eventual therapeutic administrations in HCC patients.

Erlotinib entrapped in cholesterol-depleting cyclodextrin nanoparticles shows improved antitumoral efficacy in 3D spheroid tumors of the lung and the liver.

Erlotinib (ERL), a tyrosine kinase inhibitor approved for therapeutic use in non-small cell lung cancer is further researched for eventual liver cancer treatment. However, conventional ERL has important bioavailability problems resulting from oral administration, poor solubility and gastrointestinal degradation into inactive metabolites. Alternative administration routes and nanoparticulate drug delivery systems are studied to prevent or reduce these drawbacks. In this study, ERL-loaded CD nanosphere and nanocapsule formulations capable of cholesterol depletion in resistant cancer cells were evaluated for ERL delivery. Drug loading and release profile depended largely on the surface charge of nanoparticles. Antiproliferative activity data obtained from 2D and 3D cell culture models demonstrated that polycationic ßCD nanocapsules were the most effective formulation for ERL delivery to lung and liver cancer cells. 3D tumour tumoral penetration studies further revealed that nanocapsule formulations penetrated deeper into the tumour through the multilayered cells. Furthermore, all formulations were able to extract membrane cholesterol from lung and liver cancer cell lines, indicating the induction of apoptosis and overcoming drug resistance. In conclusion, given their tumoral penetration and cell membrane cholesterol depletion abilities, amphiphilic CD nanocapsules emerge as promising alternatives to improve the safety and efficiency of ERL treatment of both liver and lung tumours.

Global omics strategies to investigate the effect of cyclodextrin nanoparticles on MCF-7 breast cancer cells.

Cyclodextrins (CD) are natural macrocyclic oligosaccharides linked by α(1,4) glycosidic bonds. Hydrophobic cavity of CDs are able to incorporate small molecules, ions, macromolecules which makes them excellent delegates for forming nanoparticulate carriers upon chemical modification to render amphiphilicity to CDs. In this study, blank 6OCaproßCD nanoparticle was prepared and administered to MCF-7 breast cancer cells. The effects of these nanoparticles on the cells were investigated in depth through biochemical and proteomic tests following 48 h of incubation. Proteomics studies revealed that apoptosis-related protein levels of hnRNP and CBX1 were increased while HDGF was not affected supporting the idea that 6OCaproßCD nanoparticles prevent cell proliferation. Gene expression studies were generally in correlation with protein levels since gene expression was significantly stimulated while protein levels were lower compared to the control group suggesting that a post-transcriptional modification must have occurred. Furthermore, 6OCaproßCD was observed to not trigger multidrug resistance as proved with RT-PCR that effectuates another exquisite characteristic of 6OCaproßCD nanoparticle as carrier of chemotherapeutic drugs. Metabolomic pathways of CD effect on MCF7 cells were elucidated with HMDB as serine biosynthesis, transmembrane transport of small molecules, metabolism of steroid hormones, estrogen biosynthesis and phospholipid biosynthesis. In conclusion, 6OCaproßCD is a promising nanoparticulate carrier for chemotherapeutic drugs with intrinsic apoptotic effect to be employed in treatment of breast cancer and further studies should be conducted in order to comprehend the exact mechanism of action.

Cellular Interaction and Tumoral Penetration Properties of Cyclodextrin Nanoparticles on 3D Breast Tumor Model.

Amphiphilic cyclodextrins are biocompatible oligosaccharides that can be used for drug delivery especially for the delivery of drugs with solubility problems thanks to their unique molecular structures. In this paper, Paclitaxel was used as a model anticancer drug to determine the inclusion complex properties of amphiphilic cyclodextrins with different surface charge. Paclitaxel-loaded cyclodextrin nanoparticles were characterized in terms of mean particle diameter, zeta potential, encapsulation efficacy, drug release profile and cell culture studies. It was determined that the nanoparticles prepared from the inclusion complex according to characterization studies have a longer release profile than the conventionally prepared nanoparticles. In order to mimic the tumor microenvironment, breast cancer cells and healthy fibroblast cells were used in 3-dimensional (3D) cell culture studies. It was determined that the activities of nanoparticles prepared by conventional methods behave differently in 2-dimensional (2D) and 3D cell cultures. In addition, it was observed that the nanoparticles prepared from the inclusion complex have a stronger anti-tumoral activity in the 3D multicellular tumor model than the drug solution. Furthermore, polycationic amphiphilic cyclodextrin nanoparticles can diffuse and penetrate through multilayer cells in a 3D tumor model, which is crucial for an eventual antitumor effect.

Development of polycationic amphiphilic cyclodextrin nanoparticles for anticancer drug delivery.

Background: Paclitaxel is a potent anticancer drug that is effective against a wide spectrum of cancers. To overcome its bioavailability problems arising from very poor aqueous solubility and tendency to recrystallize upon dilution, paclitaxel is commercially formulated with co-solvents such as Cremophor EL® that are known to cause serious side effects during chemotherapy. Amphiphilic cyclodextrins are favored oligosaccharides as drug delivery systems for anticancer drugs, having the ability to spontaneously form nanoparticles without surfactant or co-solvents. In the past few years, polycationic, amphiphilic cyclodextrins were introduced as effective agents for gene delivery in the form of nanoplexes. In this study, the potential of polycationic, amphiphilic cyclodextrin nanoparticles were evaluated in comparison to non-ionic amphiphilic cyclodextrins and core-shell type cyclodextrin nanoparticles for paclitaxel delivery to breast tumors. Pre-formulation studies were used as a basis for selecting the suitable organic solvent and surfactant concentration for the novel polycationic cyclodextrin nanoparticles. The nanoparticles were then extensively characterized with particle size distribution, polydispersity index, zeta potential, drug loading capacity, in vitro release profiles and cytotoxicity studies. Results: Paclitaxel-loaded cyclodextrin nanoparticles were obtained in the diameter range of 80-125 nm (depending on the nature of the cyclodextrin derivative) where the smallest diameter nanoparticles were obtained with polycationic (PC) ßCDC6. A strong positive charge also helped to increase the loading capacity of the nanoparticles with paclitaxel up to 60%. Interestingly, cyclodextrin nanoparticles were able to stabilize paclitaxel in aqueous solution for 30 days. All blank cyclodextrin nanoparticles were demonstrated to be non-cytotoxic against L929 mouse fibroblast cell line. In addition, paclitaxel-loaded nanoparticles have a significant anticancer effect against MCF-7 human breast cancer cell line as compared with a paclitaxel solution in DMSO. Conclusion: According to the results of this study, both amphiphilic cyclodextrin derivatives provide suitable nanometer-sized drug delivery systems for safe and efficient intravenous paclitaxel delivery for chemotherapy. In the light of these studies, it can be said that amphiphilic cyclodextrin nanoparticles of different surface charge can be considered as a promising alternative for self-assembled nanometer-sized drug carrier systems for safe and efficient chemotherapy.

Amphiphilic cyclodextrin nanoparticles.

Cyclodextrins are cyclic oligosaccharides obtained by enzymatic digestion of starch. The α-, ß- and γ- cyclodextrins contain respectively 6, 7 and 8 glucopyranose units, with primary and secondary hydroxyl groups located on the narrow and wider rims of a truncated cone shape structure. Such structure is that of a hydrophobic inner cavity with a hydrophilic outer surface allowing to interact with a wide range of molecules like ions, protein and oligonucleotides to form inclusion complexes. Many cyclodextrin applications in the pharmaceutical area have been widely described in the literature due to their low toxicity and low immunogenicity. The most important is to increase the solubility of hydrophobic drugs in water. Chemically modified cyclodextrin derivatives have been synthesized to enhance their properties and more specifically their pharmacological activity. Among these, amphiphilic derivatives were designed to build organized molecular structures, through selfassembling systems or by incorporation in lipid membranes, expected to improve the vectorization in the organism of the drug-containing cyclodextrin cavities. These derivatives can form a variety of supramolecular structures such as micelles, vesicles and nanoparticles. The purpose of this review is to summarize applications of amphiphilic cyclodextrins in different areas of drug delivery, particularly in protein and peptide drug delivery and gene delivery. The article highlights important amphiphilic cyclodextrin applications in the design of novel delivery systems like nanoparticles.

Amphiphilic Cyclodextrin Derivatives for Targeted Drug Delivery to Tumors.

Villiers has extensively studied cyclodextrins, a family of macrocyclic oligosaccharides linked by α-1,4 glycosidic bonds, in different fields since their discovery in 1891. The unique structure enabling inclusion complexation for natural cyclodextrins and cyclodextrin derivatives make them attractive for novel drug delivery systems. Cyclodextrins can be modified with long aliphatic chains to render an amphiphilic property and these different amphiphilic cyclodextrins are able to form nanoparticles without surfactants. In the literature, several different amphiphilic cyclodextrins are reported and applied to drug delivery and targeting especially to tumors. Specificly, folateconjugated amphiphilic cyclodextrin derivatives are used for active tumor targeting of poorly water soluble drugs and improve the efficacy and safety of therapeutic agents. On the other hand, effect of positive surface charge has also been under research in the recent years. Polycationic amphiphilic cyclodextrins have shown promise towards forming small complexes with negatively charged molecules such as drugs or plasmid DNA. Polycationic amphiphilic cyclodextrins enhance interaction with cell membrane due to their net positive surface charge. The scope of this review is to describe potential uses and pharmaceutical applications of tumor-targeted amphiphilic cyclodextrins, with focus on folate-conjugated cyclodextrin derivatives and polycationic cyclodextrin derivatives both studied by our group at Hacettepe University.

Cholesterol-Targeted Anticancer and Apoptotic Effects of Anionic and Polycationic Amphiphilic Cyclodextrin Nanoparticles.

Amphiphilic cyclodextrins (CDs) are biocompatible derivatives of natural CDs and are able to form nanoparticles or polyplexes spontaneously. In this study, nanoparticles prepared from nonionic (6OCaproßCD) or cationic amphiphilic CD (PC ßCDC6) were used comparatively to develop nanoparticles intended for breast cancer therapy. The characterization of these nanoparticles was performed both by in vitro and cell culture studies. Furthermore, the apoptotic and cytotoxic effects of blank amphiphilic CDs were demonstrated by various mechanistic methods including Caspase-8 activity, lipid peroxidation assay, TUNEL assay, Tali(®)-based image analysis, cholesterol assay, and gene expression studies. Blank nanoparticles exerted cytotoxicity against a variety of cancer cells (MCF-7, HeLa, HepG2, and MB49) but none to healthy cells (L929, G/G). Interestingly, blank 6OCaproßCD and blank PC ßCDC6 derivatives were found to be intrinsically effective on cell number and membrane integrity of MCF-7 cells in apoptosis studies. Further in-depth studies were performed to elucidate the selective mechanism of anticancer action in MCF-7 cells caused by these amphiphilic CDs. In conclusion, blank amphiphilic CD nanoparticles induced apoptosis through mitochondrial pathway targeted to cholesterol microdomains in cancer cell membrane.