Synchronized Codelivery of Combination Chemotherapies Intratumorally Using a Lipidic Lyotropic Liquid Crystal System.

In this work, an injectable in situ depot-forming lipidic lyotropic liquid crystal (L3C) system is developed to codeliver a precisely synchronized combination of chemotherapeutics intratumorally. The developed L3C system is composed of amphiphilic lipids and surfactants, including monoolein, phosphatidylcholine, tocopherol acetate, and d-α-tocopherol polyethylene glycol 1000 succinate. Owing to its amphiphilic nature, the developed formulation can coaccommodate both hydrophobic and hydrophilic chemotherapeutic moieties simultaneously. The study presents a proof of concept by designing a combination chemotherapy regimen in vitro and demonstrating its in vivo translation using doxorubicin and paclitaxel as model hydrophilic and hydrophobic drug moieties, respectively. The synchronized combination of the two chemotherapeutics with maximum synergistic activity was identified, coloaded in the developed L3C system at predefined stoichiometric ratios, and evaluated for antitumor efficacy in the 4T1 breast tumor model in BALB/c mice. The drug-loaded L3C formulation is a low-viscosity injectable fluid with a lamellar phase that transforms into a hexagonal mesophase depot system upon intratumoral injection. The drug-loaded depot system locally provides sustained intratumoral delivery of the chemotherapeutics combination at their precisely synchronized ratio for over a period of one month. Results demonstrate that the exposure of the tumor to the precisely synchronized intratumoral chemotherapeutics combination via the developed L3C system resulted in significantly higher antitumor activity and reduced cardiotoxicity compared to the unsynchronized combination chemotherapy or the synchronized but uncoordinated drug delivery administered by a conventional intravenous route. These findings demonstrate the potential of the developed L3C system for achieving synchronized codelivery of the chemotherapeutics combination intratumorally and improving the efficacy of combination chemotherapy.

Block Copolymer Encapsulation of Disarib, an Inhibitor of BCL2 for Improved Chemotherapeutic Potential.

A chemical inhibitor of antiapoptotic protein, BCL2, known as Disarib, suffers poor solubility in aqueous environments; thereby limiting its potential as a chemotherapeutic agent. To overcome this limitation and enhance the therapeutic efficacy of Disarib, we have employed the encapsulation of this small molecule inhibitor within P123 copolymer matrix. Micelles were synthesized using a thin-film hydration technique, and a comprehensive analysis was undertaken to evaluate the resulting micelle properties, including morphology, particle size, intermolecular interactions, encapsulation efficiency, and in vitro release characteristics. This assessment utilized various physicochemical techniques including UV spectroscopy, FTIR spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS). Disarib-loaded P123 micelle formulation denoted as P123D exhibited a well-defined particle size of approximately 29.2 nm spherical core-shell morphology. Our investigations revealed a notable encapsulation efficiency of 75%, and we observed a biphasic release pattern for the encapsulated Disarib. Furthermore, our cytotoxicity assessment of P123D micelles against mouse breast adenocarcinoma, mouse lymphoma, and human leukemic cell lines showed 40-45% increase in cytotoxicity compared with the administration of Disarib alone in the breast adenocarcinoma cell line. Enhancement in the cytotoxicity of P123D was found to be higher or limited; however, it is important to observe that the encapsulation method significantly enhanced the aqueous solubility of Disarib as it has the best solubility in dimethyl sulfoxide (DMSO) in the unencapsulated state.

An Injectable In Situ Depot-Forming Lipidic Lyotropic Liquid Crystal System for Localized Intratumoral Drug Delivery.

To address the need for localized chemotherapy against unresectable solid tumors, an injectable in situ depot-forming lipidic lyotropic liquid crystal system (L3CS) is explored that can provide spatiotemporal control over drug delivery. Although liquid crystals have been studied extensively before but their application as an injectable intratumoral depot system for locoregional chemotherapy has not been explored yet. The developed L3CS in the present study is a low-viscosity injectable fluid having a lamellar phase, which transforms into a hexagonal mesophase depot system on subcutaneous or intratumoral injection. The transformed depot system can be preprogrammed to provide tailored drug release intratumorally, over a period of one week to one month. To establish the efficacy of the developed L3CS, doxorubicin is used as a model drug. The drug release mechanism is studied in detail both in vitro and in vivo, and the efficacy of the developed system is investigated in the murine 4T1 tumor model. The direct intratumoral injection of the L3CS provided localized delivery of doxorubicin inside the tumor and restricted its access within the tumor only for a sustained period of time. This led to an over 10-fold reduction in tumor burden, reduced cardiotoxicity, and a significant increase in the median survival rate, compared to the control group. The developed L3CS thus provides an efficient strategy for localized chemotherapy against unresectable solid tumors with a great degree of spatial and temporal control over drug delivery.

Spontaneous Formation of Cationic Vesicles in Aqueous DDAB-Lecithin Mixtures for Efficient Plasmid DNA Complexation and Gene Transfection.

Cationic liposomes have become an attractive tool to deliver genes and interfering RNA into cells. Herein, we report the application of spontaneously formed cationic vesicles in mixtures of lecithin and cationic amphiphiles for efficient transfection of plasmid DNA and siRNA into cells. The average hydrodynamic diameter of the phospholipid vesicles was modulated by changing the ratio of dihexadecyldimethylammonium bromide (DDAB) to phospholipid in the vesicles. The vesicles were characterized by dynamic light scattering, ζ potential, and small-angle X-ray scattering. Depending on the ratio of DDAB to phospholipid, the average size of the vesicles can be varied in the range of 150-300 nm with a ζ potential of +40 mV. The ability of these cationic vesicles to form lipoplexes upon binding with pDNA is demonstrated by ζ potential, isothermal titration calorimetry, gel retardation, and DNase I digestion assay. The enthalpy of binding between pDNA and cationic liposome was found to be -5.7 (±0.8) kJ/mol. The cellular uptake studies of lipoplexes observed by fluorescence microscopy confirmed good transfection efficiency of DDAB liposomes in MCF-7 and HeLa cells. The fluorescent imaging analysis showed effective gene delivery and expression of green fluorescent protein. In addition, the formulation has demonstrated an ability to deliver small interfering RNA (siBRD4) for efficient gene silencing as seen by a significant decrease in BRD4 protein level in siBRD4-treated cells. Comparison of the transfection efficiency of different formulations suggests that DDAB-rich mixed phospholipid vesicles with size <200 nm are better than large size vesicles for improved endocytosis and gene expression.

Immobilization of protein on Fe3O4 nanoparticles for magnetic hyperthermia application.

We report a facile approach for the preparation of protein conjugated glutaric acid functionalized Fe3O4 magnetic nanoparticles (Pro-Glu-MNPs), having improved colloidal stability and heating efficacy. The Pro-Glu-MNPs were prepared by covalent conjugation of BSA protein onto the surface of glutaric acid functionalized Fe3O4 magnetic nanoparticles (Glu-MNPs) obtained through thermal decomposition. XRD and TEM analyses confirmed the formation of crystalline Fe3O4 nanoparticles of average size ~5 nm, whereas the conjugation of BSA protein to them was evident from XPS, FTIR, TGA, DLS and zeta-potential measurements. These Pro-Glu-MNPs showed good colloidal stability in different media (water, phosphate buffer saline, cell culture medium) and exhibited room temperature superparamagnetism with good magnetic field responsivity towards the external magnet. The induction heating studies revealed that the heating efficacy of these Pro-Glu-MNPs was strongly reliant on the particle concentration and their stabilizing media. In addition, they showed enhanced heating efficacy over Glu-MNPs as surface passivation by protein offers colloidal stability to them as well as prevents their aggregation under AC magnetic field. Further, Pro-Glu-MNPs are biocompatible towards normal cells and showed substantial cellular internalization in cancerous cells, suggesting their potential application in hyperthermia therapy.

Electrostatically bound lanreotide peptide - gold nanoparticle conjugates for enhanced uptake in SSTR2-positive cancer cells.

Lanreotide peptide (LP) has high affinity to somatostatin receptors like SSTR2 and is commonly used in the treatment of neuro-endocrine tumors. The main objective of this study is to target gold nanoparticles (AuNPs) towards SSTR2-positive cancer cells using lanreotide peptide (LP) as the targeting agent for enhanced tumor uptake and antitumor activity. pH mediated changes in the surface potential of LP and AuNP is used to prepare electrostatically bound AuNP-LP complexes. AuNP-LP complex formation was demonstrated by UV-Visible spectroscopy, surface potential, dynamic light scattering (DLS), small angle X-ray scattering and HR-TEM. Confocal microscopy and flow cytometric studies show that AuNP-LP complex has higher cellular uptake in SSTR2 expressed cancer cells (MCF-7 and AR42J) than in CHO cells. The enhanced cellular uptake of LP coated AuNPs lead to ~1.5 to 2-fold GSH depletion and enhanced ROS generation in MCF-7 cells. The preferential cytotoxicity of the AuNP-LP complex towards MCF-7 and AR42J cells, as revealed by MTT assay, is consistent with the increased cellular uptake. Our studies demonstrate that LP coated AuNP can be used as an effective platform to selectively target SSTR2 positive cancer cells for combination therapy approaches involving gold nanoparticles.

pH dependent aggregation and conformation changes of rituximab using SAXS and its comparison with the standard regulatory approach of biophysical characterization.

Development of biologics and biosimilars involves extensive physical and structural characterization, which underlines the further course of its implementation. These characterization techniques require considerable standardization and are labor intensive. It is therefore, important to have an immediate, independent and affordable characterization strategy that may meet the regulatory guidelines. In this study, we have compared the standard biophysical characterization of an anti-CD 20 antibody with characterization by small angle x ray scattering (SAXS). Aggregation of this mAb was analyzed using standard techniques like size exclusion HPLC, dynamic light scattering and sedimentation velocity - analytical ultracentrifugation, whereas structure analysis was conducted using mass spectrometry, circular dichroism spectroscopy and fluorescence spectroscopy. Our results demonstrated that the inferences about the state of mAb aggregation and its structure deduced using the standard approaches were comparable to the data interpreted using SAXS. The radius of gyration and the P(r) distribution plot obtained using the SAXS scattering data allowed analysis of aggregation and conformation of mAb via a single experiment. Thus, SAXS can be used as an independent technique to complement orthogonal analysis for determining the aggregation profile and structure of mAbs.

Altering the X-ray Scattering Contrast of Triton X-100 Micelles and Its Trapping in a Supercooled Solvent.

The structure of core-shell micelles formed by nonionic surfactant Triton X-100 (TX-100) in a supercooled glucose-urea melt is investigated by contrast variation small-angle X-ray scattering (SAXS), small angle neutron scattering (SANS), and HR-TEM. Cooling a molten mixture of glucose-urea (weight ratio of 3:2) to room temperature yields a supercooled solvent without crystallization that can be used for trapping micelles of TX-100. By use of a combination of water and glucose-urea mixture at different proportions as solvent for micellization, the scattering length density (SLD) of the solvent can be tuned to match the shell contrast of the micelles. A systematic analysis of SAXS and SANS data with different SLD of solvent permits a quantitative evaluation of electron density profile of micelles in different matrices. The core of TX-100 micelles shows significant swelling in glucose-urea melt, as compared to that in water. The dimension and morphology of micelles were evaluated by scattering techniques and HR-TEM. Dynamic light scattering (DLS) studies suggest that, unlike micelles in water, the diffusion of micelles in supercooled glucose-urea melt decreased by several orders of magnitude.

High antimicrobial photodynamic activity of photosensitizer encapsulated dual-functional metallocatanionic vesicles against drug-resistant bacteria S. aureus.

Developments in the field of photodynamic therapy (PDT) are being made by investigating appropriate photosensitizers (PSs) and enhancing the penetration effect of light by developing new nano-carriers. So, to boost the PDT effect, in the present work, new metallocatanionic vesicles were fabricated by a convenient, efficient, green and inexpensive method to encapsulate PSs and evaluate their antimicrobial PDT against the drug-resistant bacterium Staphylococcus aureus. They were prepared from a combination of a double-chained copper-based cationic metallosurfactant (CuCPCII) and an anionic surfactant sodium bis(2-ethylhexyl)sulfosuccinate (Aerosol OT or AOT). The surface charge, structure and ability to encapsulate oppositely charged photosensitizers are some crucial factors that need to be controlled for their effective utilization in PDT. In this approach, two of the fractions, one each from a cationic rich and anionic rich side, were selected to encapsulate cationic (methylene blue; MB) and anionic (rose bengal (RB)) PSs after characterization by SAXS, AFM, FESEM, DLS, and zeta-potential, and conductivity measurements. Afterwards, PDT was performed on S. aureus (a multidrug-resistant bacterium) by the colony forming unit (CFU) method using PS encapsulated metallocatanionic vesicles that demonstrated high bactericidal activity by using visible light (532 nm) and facilitated the generation of singlet oxygen. The singlet oxygen generation capability of both the PSs was enhanced under irradiation when encapsulated in metallocatanionic vesicles because the presence of metal accelerated the intersystem crossing of triplet oxygen to singlet oxygen. Furthermore, these studies reveal that the metallocatanionic vesicles have dual functionality i.e. encapsulate PSs and even show dark toxicity against S. aureus. To study the killing of S. aureus, bacterial DNA was extracted and its interactions and conformational changes in the presence of metallocatanionic vesicles were analyzed via., UV-Visible, and circular dichroism (CD) spectroscopy. Comet assay (single-cell gel-electrophoresis) demonstrated the DNA damage after PDT treatment in an individual cell. The bacterial DNA damage was more with the metallosurfactant rich 70 : 30 fraction than with the 30 : 70 fraction, in combination with RB under irradiation. This work provides a new metal hybrid smart material that possesses dual functionality and is prepared by an easy, economical and feasible procedure which resulted in enhanced PDT against a drug-resistant bacterium, thus, providing an alternative for antibacterial therapy.

pH-Labile Magnetic Nanocarriers for Intracellular Drug Delivery to Tumor Cells.

We report the development of pH-labile ascorbic acid-coated magnetic nanocarriers (AMNCs) for effective delivery of the anticancer drug doxorubicin hydrochloride (DOX) to tumor cells. The uniqueness of this drug delivery system lies in the covalent conjugation of DOX through carbamate and hydrazone bonds, resulting in a slow and sustained drug release profile at different environmental acidities. X-ray diffraction and transmission electron microscopy analyses reveal the formation of crystalline single-phase Fe3O4 nanoparticles with an average size of 10 nm. The changes in the interfacial characteristics of the nanocarriers and the presence of organic coatings are probed by infrared spectroscopy, dynamic light scattering, zeta potential, and thermogravimetric measurements. AMNCs show high colloidal stability in aqueous and cell culture media and possess good magnetic field responsivity and protein resistance characteristics. The drug-loaded nanocarriers exhibited sustained pH-triggered release of drug molecules in acidic mediums, substantial cellular internalization, and significant toxicity toward the proliferation of mouse skin fibrosarcoma (WEHI-164), human breast cancer (MCF-7), and human lung cancer (A549) cells. However, it showed significantly lower toxicity in human normal lung (WI26VA) cells. Overall, these results suggest a pH-sensitive drug release of nanoformulations, which showed selective toxicity to tumor than normal cells.

Discerning the Structure Factor of Charged Micelles in Water and Supercooled Solvent by Contrast Variation X-ray Scattering.

Sodium dodecyl sulfate (SDS) is a well-known anionic surfactant that forms micelles in various solvents including supercooled sugar-urea melt. Here, we explore the application of contrast variation small-angle X-ray scattering (SAXS) in discerning the structure and interactions of SDS micelles in aqueous solution and in a room-temperature supercooled solvent. The SAXS patterns can be analyzed in terms of a core-shell ellipsoid model. For aqueous SDS micelles, at low volume fractions, the features due to intermicellar interaction, S(q), in the SAXS pattern are poorly resolved because of the prominent contribution from shell scattering. Increasing the electron density of the solvent by the addition of the urea or fructose-urea mixture (at a weight ratio of 6:4) permits the systematic variation of shell scattering without influencing the structure drastically. For a 10% solution of SDS in water, the contribution from the shell can be completely masked by the addition of 40% urea or fructose-urea mixture. The fructose-urea mixture is a preferred additive as it can vary the scattering length density over a wide range and serves as a matrix to form supercooled micelles. The structural parameters of micelles in supercooled fructose-urea melt are obtained from contrast variation SAXS, small-angle neutron scattering, and high-resolution transmission electron microscopy.

Directing Amphiphilic Self-Assembly: From Microstructure Control to Interfacial Engineering.

The self-assembly of small molecules into complex nanoscale structures is driven by the interplay of various noncovalent interactions. It has now become evident that by maneuvering this intermolecular interaction the geometry and interfacial properties of several nanoscale objects can be tamed. In particular, diverse structures such as spheres, rods, worms, ribbons, and vesicles can be produced by tuning the packing of molecules in the aggregate. Stimuli-sensitive assemblies that can reversibly associate or dissociate in response to environmental changes have been fabricated as model systems for the self-regulated drug delivery vehicle. Surface passivation of inorganic materials can be achieved by the selective organization of molecules at the interface. Such surface functionalization of inorganic materials by organic counterparts provides kinetic stability in biological media and permits the selective binding of active ingredients. Advances made in the area of molecular self-assembly and factors governing such association processes have made it possible to control the interfacial properties and microstructure of nanoscale materials.

Cationic double chained metallosurfactants: synthesis, aggregation, cytotoxicity, antimicrobial activity and their impact on the structure of bovine serum albumin.

Bovine serum albumin (BSA) is one of the most copious and significant blood proteins with dynamic structure. The understanding of the structural functionality of BSA and its interaction with metal ions is desired for various biological functions. Herein, three different metallosurfactants containing different transition metals and the same hydrophobic tail were engaged to investigate the structural transition of BSA. The metallosurfactants have been prepared by a combination of metal ions (M = Fe, Co and Ni) with cetylpyridinium chloride surfactant via the ligand insertion method and were characterized by elemental, FTIR, 1H-NMR, and thermogravimetric analysis (TGA). The obtained results reveal that insertion of a metal ion perturbs the aggregation behavior of the surfactant. Incorporation of a metal-ion has been found to decrease the CMC value of the surfactant, which has been supported by conductivity, surface tension and small angle X-ray scattering (SAXS). These metallosurfactants were employed to study the interaction and binding mechanism of BSA under physiological conditions. SDS-PAGE analysis points out a weak effect of metallosurfactants on the primary structure of BSA, whereas CD spectra implied a significant change in secondary structure with the decreased α-helical content of BSA. Fluorescence spectroscopy indicates the effect of metallosurfactants on the tertiary structure of BSA, whereas absorption spectra demonstrated static quenching with a blue shift in the presence of metallosurfactants. Moreover, unfolding of BSA in the presence of metallosurfactants has also been confirmed by SAXS studies. The overall results indicate that insertion of the metal ion into the framework of the surfactant structure enhances its protein binding/folding/unfolding abilities, which would be helpful in clinical as well as in life sciences.

Investigating the structural integrity of Bovine serum albumin in presence of newly synthesized metallosurfactants.

Bovine Serum Albumin is major transport protein and is often used as a drug carrier in body organs. Knowledge of its binding with metallosurfactant can significantly influence the biodistribution of metallodrugs. Current work demonstrated a facile method to prepare four different double chained metallosurfactants containing Fe, Co, Ni and Cu as part of their counter ion. The as-synthesized metallosurfactants were characterized using FTIR, AAS, TGA and XRD in solid form. The aggregation of these metallosurfactants in aqueous medium was investigated through conductivity, surface tension and SAXS. Further, we have investigated their binding with BSA through different analytical methods The effect of concentration of metallosurfactants on the primary and secondary structure of BSA was further examined by SDS-PAGE and Circular dichroism, respetively. It is found that at premicellar concentration, the primary structure of BSA was not affected but the secondary structure i.e. α-helical structure of BSA was altered as shown by circular dichroism. Interestingly, post micellar concentration of metallosurfactants shows the pronounced effect on the primary and secondary structure of BSA. SAXS study also supports the fact of unfolding of protein and its wrapping around the micelles. Zeta potential describes the electrical charge and stability of the protein in the presence of different concentration of metallosurfactant. Along with, it was found that presence of protein delays the aggregation behavior of metallosurfactant, as a sign of binding of BSA with metallosurfactant.

Fabrication of metalosomes (metal containing cationic liposomes) using single chain surfactants as a precursor via formation of inorganic organic hybrids.

Among the self-assembled forms of surfactants, vesicles/liposomes are a highly promising and interesting feature of surfactants, which are usually formed from water insoluble surfactants. Herein, we demonstrate the formation of liposomes from single-chain cationic surfactants with the help of metals as a part of the counter ion, and these metal embedded liposomes are termed as metalosomes. It is a noteworthy advancement in the area of self-assembled molecular structures since we report the preparation of metal embedded liposomes (metalosomes) from a water soluble single chain cationic surfactant, which is otherwise a property or an arrangement made by double tailed surfactants, or more precisely lipids that are poorly water soluble. We can use this method for various cationic surfactants and metal combinations and the studies are still in process. However, this preliminary report on manganese-based surfactants depicts the successful formation of cationic metalosomes (with/without cholesterol), and the formation, structure and size has been verified using TEM, FE-SEM, DLS XRD and SAXS. The comparison of metalosomes with reverse vesicles in different solvents further gave an insight of microstructure and solvent environment effects on the self-assembly of metallosurfactants. In addition, we have also evaluated the encapsulation ability of metalosomes for fluorescein dye. High encapsulation efficiency of Mn-somes makes them promising candidates for several applications, particularly because of its water solubility.