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8-11-2020

Heat Shock Protein 90 ()-Inhibitor-Luminespib-Loaded- Protein-Based Nanoformulation for Cancer Therapy

Ankit K. Rochani Thomas Jefferson University

Sivakumar Balasubramanian Toyo University

Aswathy Ravindran Girija Toyo University

Toru Maekawa Toyo University

FGaganollow this Kaushal, and additional PhD works at: https://jdc.jefferson.edu/pharmacyfp Thomas Jefferson University Part of the Other Pharmacy and Pharmaceutical Sciences Commons Let us know how access to this document benefits ouy See next page for additional authors

Recommended Citation Rochani, Ankit K.; Balasubramanian, Sivakumar; Ravindran Girija, Aswathy; Maekawa, Toru; Kaushal, PhD, Gagan; and Kumar, D Sakthi, " 90 (Hsp90)-Inhibitor- Luminespib-Loaded-Protein-Based Nanoformulation for Cancer Therapy" (2020). College of Pharmacy Faculty Papers. Paper 43. https://jdc.jefferson.edu/pharmacyfp/43

This Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital Commons is a service of Thomas Jefferson University's Center for Teaching and Learning (CTL). The Commons is a showcase for Jefferson books and journals, peer-reviewed scholarly publications, unique historical collections from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers and interested readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been accepted for inclusion in College of Pharmacy Faculty Papers by an authorized administrator of the Jefferson Digital Commons. For more information, please contact: [email protected]. Authors Ankit K. Rochani; Sivakumar Balasubramanian; Aswathy Ravindran Girija; Toru Maekawa; Gagan Kaushal, PhD; and D Sakthi Kumar

This article is available at Jefferson Digital Commons: https://jdc.jefferson.edu/pharmacyfp/43 polymers

Article Heat Shock Protein 90 (Hsp90)-Inhibitor-Luminespib- Loaded-Protein-Based Nanoformulation for Cancer Therapy

Ankit K. Rochani 1,2, Sivakumar Balasubramanian 1, Aswathy Ravindran Girija 1, Toru Maekawa 1, Gagan Kaushal 2 and D. Sakthi Kumar 1,*

1 Bio Nano Electronics Research Centre, Graduate School of Interdisciplinary New Science, Toyo University, Saitama 350-8585, Japan; ankit.rochani@jefferson.edu (A.K.R.); [email protected] (S.B.); [email protected] (A.R.G.); [email protected] (T.M.) 2 Jefferson College of Pharmacy, Thomas Jefferson University, Philadelphia, PA 19107, USA; gagan.kaushal@jefferson.edu * Correspondence: [email protected]; Tel.: +81-49-239-1636 or +81-49-239-1375 or +81-49-239-1640; Fax: +81-49-234-2502  Received: 8 July 2020; Accepted: 7 August 2020; Published: 11 August 2020 

Abstract: Drugs targeting heat shock protein 90 (Hsp90) have been extensively explored for their anticancer potential in advanced clinical trials. Nanoformulations have been an important drug delivery platform for the anticancer molecules like Hsp90 inhibitors. It has been reported that bovine serum albumin (BSA) nanoparticles (NPs) serve as carriers for anticancer drugs, which have been extensively explored for their therapeutic efficacy against cancers. Luminespib (also known as NVP-AUY922) is a new generation Hsp90 inhibitor that was introduced recently. It is one of the most studied Hsp90 inhibitors for a variety of cancers in Phase I and II clinical trials and is similar to its predecessors such as the ansamycin class of molecules. To our knowledge, nanoformulations for luminespib remain unexplored for their anticancer potential. In the present study, we developed aqueous dispensable BSA NPs for controlled delivery of luminespib. The luminespib-loaded BSA NPs were characterized by SEM, TEM, FTIR, XPS, UV-visible spectroscopy and fluorescence spectroscopy. The results suggest that luminespib interacts by non-covalent reversible interactions with BSA to form drug-loaded BSA NPs (DNPs). Our in vitro evaluations suggest that DNP-based aqueous nanoformulations can be used in both pancreatic (MIA PaCa-2) and breast (MCF-7) cancer therapy.

Keywords: BSA nanoparticles; Hsp90; luminespib (NVP-AUY922); pancreatic cancer; breast cancer

1. Introduction The development of drugs against breast and pancreatic cancer is a major challenge due to various back channels, such as drug efflux mechanism by p-glycoproteins (p-gp), development of drug resistance to human epidermal growth factor 2 (HER2) and overexpression of heat shock proteins (HSPs). Newer strategies such as mono or combination therapies that target multiple cellular pathways of cancer cells are being developed to fight against these complexities of breast and pancreatic cancer [1–3]. Studies suggests that development of drug-targeting HSPs is one of the most promising ways to fight a variety of cancers [4]. The microenvironment of cancer cells is extremely stressful due to acidic pH, oxidative stress, nutritional stress, phototoxic stress, hypoxic and other stress conditions. This turbulent cellular microenvironment serves as a hallmark for cancer. It has been shown that heat shock protein 90 (Hsp90) is overexpressed in cancer cells in order to buffer such stressful conditions and provide cyto-protective function to them [4–6]. Hsp90 is an important molecular , which has thousands of client

Polymers 2020, 12, 1798; doi:10.3390/polym12081798 www.mdpi.com/journal/polymers Polymers 2020, 12, 1798 2 of 17 proteins that play an important roles in the growth promotion and the survival of cancer cells under stress conditions [7]. Hence, Hsp90 works as a central hub for survival of cancer cells by interacting with a large number of unfolded or damaged essential proteins and helps in their folding by forming a multichaperone complex. As a result, inhibition of Hsp90 can hinder a large number of essential cellular pathways for cancer cells, which ultimately leads to cell death. An ansamycin analog-17-allylamino 17-demethoxygeldanamycin (17AAG)—was the first Hsp90 inhibitor that entered clinical trial against cancer by Bristol Myers Squibb. There are around fifteen molecules that have been explored as single or combination therapies. Most Hsp90 inhibitors acts by binding to the N-terminal domain that causes inhibition of the protein-folding pathway followed by activation of proteasomal degradation of client protein [8]. Being one of the most promising cancer drug targets, Hsp90 inhibitors were explored for their efficacy against breast cancer therapy. It was reported that 17AAG proved to be effective in a phase II trial for HER2 +ve breast cancer therapy [9]. Other Hsp90 inhibitors, like 17-Dimethylaminoethylamino- 17-demethoxygeldanamycin (17DMAG), 17-Allylamino-17- Demethoxygeldanamycin Hydroquinone Hydrochloride (IPI-504), have also shown promising results towards breast cancer, pancreatic cancer and various forms of cancer treatment in single or combination therapy. This indicates that Hsp90 inhibitors can be effective therapeutic molecules against multiple cancer types. Various parent structures like , purine analogs and others are being pursued in order to identify newer derivatives as anticancer molecules, which offers improved binding affinities towards Hsp90 proteins and are effective against various phenotypes of cancers compared to first generation ansamycin analogs [10,11]. Luminespib was recently introduced in the quest for finding a newer potent Hsp90 inhibitor. It is an iso-oxazole resorcinol-based synthetic Hsp90 inhibitor (as shown in Figure1a) that was discovered with an intention to develop newer and better anticancer molecules [12]. It is was explored for its broad-spectrum anticancer activity in >10 clinical trials (clinicaltrials.gov), including estrogen receptor (ER) +ve and herceptine (HER) +ve breast cancer conditions [13,14]. The molecule was also found to be effective towards pancreatic cancer (MIA PaCa-2) cell line [15]. Hence, we believe that this single molecule may be useful for in multiple cancer types. Although it is a clinically explored experimental new drug molecule, luminespib remains unexplored for developing controlled delivery nanoformulations. The only formulation used for luminespib in a preclinical breast cancer model was in 60-mM lactic acid or 2.5% ethanol, 20% 50-mM tartaric acid and 77% (5% glucose in water containing 1% Tween-80) vol/vol [12,16]. Infusion of the complex formulation comprising ethanol, tartaric acid and lactic acid may lead to dose dependent toxicity. The molecule was explored in early Phase I and Phase II clinical trials against breast cancer, non-small cell lung cancer, refractory multiple myeloma, advanced solid tumors and others [17–19]. We believe that the development of water dispensable nanoformulations of luminespib should help to increase patient compliance. A number of polymeric and nonpolymeric nanocarriers have been extensively explored for their potential drug formulation applications [20,21]. Polymeric nanoformulations have played an important role in developing successful nanoparticle (NP)-based anticancer therapy [7]. Polymeric nanoformulations have been used in developing novel aqueous formulation for hydrophobic Hsp90 inhibitors like 17AAG. Our lab had developed hydrophilic polymeric nanoformulations as 2-in-1 iron oxide (MNP) and 17AAG-loaded polymeric nanoformulation with the aim of combining the cytotoxicity property of 17AAG with magnetic hyperthermia in the same formulation [22]. Recently, there is a constant demand in the ideas of using various biopolymers to make better nanoformulations. Protein-based carriers such as serum albumin have shown an intriguing potential to be utilized in nanoformulation [23,24]. Serum albumin is one of the most abundant proteins in the circulating system of a wide variety of organisms. The two most common types of albumin that are used in biomedical applications; (a) bovine serum albumin (BSA) and (b) human serum albumin (HSA). BSA shares nearly 76% sequence homology with HSA [25]. Hence, BSA is the most commonly used model protein for drug interaction studies and nanodrug delivery vehicle. Serum albumin is a major molecule in the plasma of animals, and it accounts for nearly 60% of total protein (with a concentration of 42 g Polymers 2020, 12, x FOR PEER REVIEW 6 of 17 Polymers 2020, 12, 1798 3 of 17

3. Results and Discussion 3 dm− ). It consists of a single chain of 582 amino acids divided into three linearly arranged domains (I–III)3.1. Synthesis, [26]. The Particle interaction Size Characterizat of anticancerion drugs and Encapsulation with BSA has of been BSA extensively Luminespib explored NPs for developing novelSynthesis formulations. of DNPs Nab-paclitaxel was carried (Abraxane)out using a isdesolvation the first albumin-stabilized method wherein ethanol nanoformulation acts as an toanti- be solventapproved as byshown Food in and Figure Drug 1c Administration [32,36]. Our dynamic (FDA) inlight 2005 scattering for its effi (DLS)cacy instudies the treatment showed ofthat breast the averagecancer [ 27particle]. It was size also of DNPs approved was byaround FDA 222.43 for metastatic ± 1.150 nm pancreatic as shown cancer in Table and 1 non-smalland Figure cell 2a. lungThe zetacancer. potential BSA–dextran–folic of the nanoformulation acid–doxorubicin was found nanoconjugate to be −30.63 ± also 1.365 showed mV with effi acacy poly in dispersity murine ascites index ofhepatoma 0.133 ± 0.014 H22 as tumor-bearing shown in Figure mice 2b [28 and]. Furthermore,Table 1, respectively. gemcitabine We believe and folate that gemcitabine-loadedlow poly dispersity andnanoconjugates particle size showed of around effi cacy222 nm against may pancreaticbe attribut anded to MCF-7 the ethanol breast as cancer desolvating cell lines agent [29]. and Albumin pH 8 usednanocarriers during the for synthesis 17AAG were of the also nanoparticles explored. It [37,38]. was found Moreover, that nab-17AAG our synthesized with an nanoformulation average size of shows210 nm similar was shown DLS and to have particle considerable size distribution anticancer characteristics activity towards as shown MX-1 in breastpreviously cancer reported cell line cases [30]. ofThe BSA study NPs provided [32]. The positive particle evidence size and that spherica the nabl morphology formulation ofof Hsp90the synthesized inhibitor couldDNPs improve were also its confirmedefficacy, safety by SEM and and stability TEM as in shown comparison in Figure to their 2c,d. surfactant Further, the and drug solvent-based entrapment formulationsstudies show that [30]. theBased DNPs on the has success encapsulation of earlier efficiency studies, we of have 48.22 explored ± 1.948% the and possibility drug-loading to develop of 4.28 BSA ± nanoparticles1.94 [39]. To evaluateconsisting and of luminespibconfirm the and probable investigated drug–protein its biocompatibility interaction mechanism and efficacy we towards performed MCF-7 molecular and MIA simulationsPaCa-2 cell lines.and fluorescent quenching studies.

Figure 1. (a) and (b) Structure of luminespib and bovine serum albumin (BSA) (protein databank Figure 1. (a,b) Structure of luminespib and bovine serum albumin (BSA) (protein databank (PDB): (PDB): 3V03), respectively; (c) schematics of synthesis and anticancer efficacy of drug-loaded BSA 3V03), respectively; (c) schematics of synthesis and anticancer efficacy of drug-loaded BSA nanoparticles nanoparticles(NPs) towards (NPs) cancer towards cell lines cancer in comparison cell lines in to comparison native or blank to native BSA NPs.or blank BSA NPs.

2. Materials andTable Methods 1. Physical-chemical characterization of drug-loaded BSA NPs (DNPs). BSA was obtained from Sigma-AldrichParameters (Tokyo, Japan), ethanol (99%)Values was procured from Wako (Tokyo, Japan); luminespibZ-average was particle purchased size from(nm) LC laboratories (Woburn, 222.433 ± MA,1.150 USA). Thin-layer chromatography (TLC)Zeta plates potential were obtainedaverage (mV) from Merck Millipore (Burlington,−30.63 ± 1.365 MA, USA). Trypan blue, trypsin EDTA (0.025%),Poly dispersity alamar blueindex cytotoxicity (PDI) kit and Dulbecco’s 0.133 modified ± 0.014 Eagle’s medium (DMEM) were procuredEncapsulation from Invitrogen efficiency (Tokyo, (%) Japan). 48.22 ± 1.948 Drug loading 4.28 ± 1.94

Polymers 2020, 12, 1798 4 of 17

2.1. Synthesis of BSA-Luminespib Nanoconjugates The synthesis of luminespib (drug) BSA nanoconjugates was achieved using desolvation method [31,32]. BSA protein (100 mg) was dissolved in distilled water and the pH was adjusted to around 8.0. The basic pH adjustments were performed using 0.1-M NaOH. To 10 mg of luminespib, 2 mL of absolute ethanol was added to dissolve the drug, and the (ethanolic-drug) solution was dropwise added to BSA (0.5 mL) solution at the rate of 0.5 mL/min using a syringe pump. Slow addition of drug containing ethanol solution causes formation of white suspension that indicates formation of drug–BSA conjugates. Native BSA NPs were prepared by controlled addition of ethanol to BSA solution. These drug–BSA NPs and native BSA NPs were subjected to crosslinking with 8% glutaraldehyde solution at room temperature for 4 to 5 h. The cross-linked drug-BSA nanoconjugates were subjected to washing with distilled water and centrifuged at 15,000 rpm. The collected pellet was freeze-dried and reconstituted in phosphate buffer saline (7.4 pH, PBS) for further studies.

2.2. Luminespib BSA Particle Characterization Studies Specific quantity of BSA-drug nanoconjugates was filtered and analyzed for particle size, zeta potential and poly-dispersity index determination using Malvern NanoZS analyzer. The data were recorded using Zetasizer (version 2.0, Malvern Panalytical, Tokyo, Japan). All the experiments were performed in triplicates and the data were expressed as standard deviation. Surface morphology characteristics of nanoparticles were determined using scanning electron (SEM) and Transmission electron microscopy (TEM). For SEM, the sample was dropped on glass substrate, vacuum dried and coated with platinum for taking images using SEM 6600 (Hitachi, Tokyo, Japan). DNP sample was dropped on TEM hydrophilic copper grid and images were taken using JEM-2100 (Jeol, Tokyo, Japan), at 160 kV.

2.3. In Silico Molecular Docking Docking calculations were carried out using Docking Server [33]. The crystal structure of BSA was downloaded from protein databank (PDB), accession number 3V03. The structure of the ligand (luminespib) with PubChem ID 135539077 was taken for carrying out the docking simulations. PM-6 partial calculations method was used for both ligand and protein. Further, the geometry for ligand was performed using MMFF94. Nonpolar hydrogen atoms were merged, and rotatable bonds were defined. Essential hydrogen atoms, Kollman united atom type charges, and solvation parameters were added with the help of AutoDock tools [34]. Affinity (grid) maps of 90 60 80 Å grid points and 0.375 Å × × spacing were generated using the Autogrid program (Virtua Drug, Budapest, Hungary and Scripps Research Institute, La Jolla, CA, USA). AutoDock parameter set and distance-dependent dielectric functions were used in the calculation of the van der Waals and the electrostatic terms, respectively. Docking simulations were performed using the Lamarckian genetic algorithm (LGA) and the Solis & Wets local search method used by DockingServer GUI [35]. Initial position, orientation and torsions of the ligand molecules were set randomly. All rotatable torsions were released during docking. Each docking experiment was derived from 100 different runs that were set to terminate after a maximum of 2,500,000 energy evaluations. The population size was set to 150. During the search, a translational step of 0.2 Å, and quaternion and torsion steps of 5 were applied. For analysis, docking results were ranked based on free energy of binding (kcal/mol), number of hydrogen bonds, polar interactions and frequency of probable binding sites. The frequency shows the percentage of the local searches with similar geometry having root mean square tolerance (rmstol) of 2 Å. The docked structures with lowest binding free energy, with maximum number of polar and hydrogen bond interactions, and with frequency of 10% or more were used for prediction of probable binding configuration. The analyzed docking file in.pdb format was downloaded and analyzed using Discovery Studio Visualizer 4.0 (Biovia, Tokyo, Japan). Polymers 2020, 12, 1798 5 of 17

2.4. Luminespib-BSA Interaction Studies

2.4.1. Fluorescence Quenching Studies for Drug BSA Complex Fluorescence spectroscopy measurements were carried out for a fixed concentration of BSA 12.5 µM. The drug concentration was varied from 0.0 to 12.5 µM at room temperature. Various concentrations of drug were made in 30% ethanolic PBS solution at pH 7.4. Fluorescence spectra were measured using FP6500 (Jasco, Tokyo, Japan), Jasco instrument. The excitation were kept as 280 nm. Further, at the same instrument settings we also checked the quench in fluorescence spectra for BSA–drug nanoconjugates in comparison to native BSA particle in PBS (pH 7.4) at 1 mg/mL concentration.

2.4.2. UV-Vis Absorption Studies To determine the presence of drug in the BSA NPs; we performed UV-vis. absorption studies for 1 mg/mL concentration of drug–BSA conjugates (DNPs) dispersion in PBS (pH 7.4). Here, the UV spectrum was also recorded for the plain BSA nanoparticle and free drug as control.

2.4.3. Thin-Layer Chromatography (TLC) To understand the drug interaction with BSA and formation of nab-luminespib nanoformulations, we performed TLC measurements. We spotted 10 µL of 1 mg/mL concentration of DNPs in deionized water on TLC plates and compared them to blank BSA NPs and native drug as controls. We used chloroform, methanol and concentrated NH4OH (80:20:1) as mobile phase. Rf value was calculated for spots after checking TLC plates under UV illumination.

2.4.4. X-ray Photoelectron Spectroscopy Studies (XPS) XPS measurement was performed using Phi Quantes XPS system (Ulvac-Phi, Kanagawa, Japan). Samples were mounted on glass slide and subjected to recording for individual spectral recordings for carbon, nitrogen, oxygen and wide scan. All the measurements were made using aluminum X-ray at 55-pass energy. The XPS data were analyzed using Multipack software (Ulvac-Phi, Kanagawa, Japan).

2.5. Encapsulation and Drug Loading Evaluation In our study, 1 mg of BSA-drug nanoconjugates was taken and dispersed in 1 mL of ethanol and subjected to quantitation of luminespib at 310 nm (DU® 730 UV Spectrometer by Beckman Coulter, Tokyo, Japan). Drug encapsulation and drug-loading efficiency studies were performed in triplicates using the following Equations (1) and (2):

Amount o f Luminespib in nanoparticles Encapsulation e f f iciency (%) = 100 (1) Total weight o f Luminespib used f or synthesis ×

Amount o f Luminespib in nanoparticles Drug loading e f f iciency (%) = 100 (2) Gross weight o f nanoparticles ×

2.6. Stability of DNPs To study the stability of DNPs at ambient room temperature, nanoparticles were stored at 25 ◦C in glass vial. Samples were drawn to check the stability of formulation for up to seven days. Here, the particle size distribution and SEM image were used as quantitative and qualitative checkpoints, respectively.

2.7. In Vitro Drug Release for DNPs In our study, 20 mg of nanoparticles were taken and dispersed in 20 mL of deionized water. The solutions were distributed in Eppendorf tubes such that each tube consisted of 1 mg/mL concentration. All the tubes were kept in a shaking incubator at 37 ◦C. At predetermined time Polymers 2020, 12, 1798 6 of 17 intervals supernatant was taken and subjected to UV absorbance reading at 310 nm for determination of quantity of drug released. All the measurements were performed in triplicates and data were plotted as an average of the drug released at specific time points.

2.8. Cell Culture and In Vitro Cytotoxicity Studies To check the cytotoxicity, we carried out culturing of MCF-7 and MIA PaCa-2 cancer cell lines. We also cultured normal mouse fibroblast cell line (L929) as control. MCF-7 and L929 cell lines were cultured in DMEM media consisting of 10% FBS and 5% penicillin/streptomycin (100 units per mL). MIA PaCa-2 was cultured in RPMI media with 10% FBS and 5% penicillin/streptomycin (100 units per mL). All the cells were incubated at 37 ◦C in a humidified CO2 environment. The confluent T-25 plates were sub cultured every third day. MCF-7, MIA PaCa-2 and L929 cells were seeded in 96-well plates with a cell density of 5000 cells per well and cytotoxicity assay was performed with the synthesized nanoformulations using alamar blue dye. After 24 h of incubation in 96-well plates, cell lines were treated with DNPs in concentration dependent manner from 100 to 1000 µg/mL. The treated plates were subjected to measurement of fluorescence at excitation and emission wavelength as 580 and 530 nm, respectively. The measurements were made using Power scan HT, Micro plate reader, Dainippon Sumitomo Pharma, Osaka, Japan. The cell viability measurements were made for 48 h. We used Equation (3) for measurement of% cell viability. All the experiments were performed in triplicates and subjected to Student’s t-test for statistical significance. Asample Cell viability (%) = 100 (3) Acontrol ×

3. Results and Discussion

3.1. Synthesis, Particle Size Characterization and Encapsulation of BSA Luminespib NPs Synthesis of DNPs was carried out using a desolvation method wherein ethanol acts as an anti-solvent as shown in Figure1c [ 32,36]. Our dynamic light scattering (DLS) studies showed that the average particle size of DNPs was around 222.43 1.150 nm as shown in Table1 and Figure2a. The ± zeta potential of the nanoformulation was found to be 30.63 1.365 mV with a poly dispersity index − ± of 0.133 0.014 as shown in Figure2b and Table1, respectively. We believe that low poly dispersity ± and particle size of around 222 nm may be attributed to the ethanol as desolvating agent and pH 8 used during the synthesis of the nanoparticles [37,38]. Moreover, our synthesized nanoformulation shows similar DLS and particle size distribution characteristics as shown in previously reported cases of BSA NPs [32]. The particle size and spherical morphology of the synthesized DNPs were also confirmed by SEM and TEM as shown in Figure2c,d. Further, the drug entrapment studies show that the DNPs has encapsulation efficiency of 48.22 1.948% and drug-loading of 4.28 1.94 [39]. To evaluate and ± ± confirm the probable drug–protein interaction mechanism we performed molecular simulations and fluorescent quenching studies.

Table 1. Physical-chemical characterization of drug-loaded BSA NPs (DNPs).

Parameters Values Z-average particle size (nm) 222.433 1.150 ± Zeta potential average (mV) 30.63 1.365 − ± Poly dispersity index (PDI) 0.133 0.014 ± Encapsulation efficiency (%) 48.22 1.948 ± Drug loading 4.28 1.94 ± Polymers 2020, 12, 1798 7 of 17 Polymers 2020, 12, x FOR PEER REVIEW 7 of 17

Figure 2. (a,b) Particle size distribution between 78 to 500 nm and zeta-potential distribution of −11.8 Figure 2. (a,b) Particle size distribution between 78 to 500 nm and zeta-potential distribution of 11.8 − toto −48.148.1 mV. TheThe average average particle particle size size (obtained (obtained from from DLS) DLS) was was found found to be aroundto be around 222 nm 222 and nm average and − averagezeta potential zeta potential as around as around30 mV; (−c30,d) mV; SEM ( andc,d) TEMSEM imagesand TEM of theimages synthesized of the synthesized DNPs, respectively. DNPs, − respectively.SEM images SEM shows images particle shows various particle sizes various around sizes ~200 around nm. TEM ~200 images nm. TEM confirms images small confirms particle small size particle~100 to 150size nm.~100 The to 150 DNP nm. images The DNP from images SEM and from TEM SEM confirmed and TEM the confirmed observation the from observation DLS. from DLS. 3.2. BSA Luminespib Interaction Studies 3.2. BSA Luminespib Interaction Studies Synthesis of BSA luminespib NPs depends on the interaction of luminespib with BSA at the structuralSynthesis level. of ToBSA evaluate luminespib this asNPs a first depends step, weon th performede interaction luminespib of luminespib docking with studies BSA toat findthe structuralthe probable level. binding To evaluate site. Ourthis resultas a first indicates step, we that performed Gibbs free luminespib energy of docking drug binding studies to to BSA find wasthe probable10.48 kcal binding/mol with site. frequency Our result of indicates 50% and that predicted Gibbs inhibitionfree energy (K of) as drug 20.64 binding nM as shownto BSA inwas Figure −10.483a. − i kcal/molThe model with shows frequency that luminespib of 50% and may predicted form relatively inhibition more (Ki) as stable 20.64 drug-BSA nM as shown complex in Figure compared 3a. The to modelpreviously shows reported that luminespib cases of drug-BSA may form interaction relatively due more to relativelystable drug-BSA low binding complex free energycompared found to previouslyin the range reported of 4.35 cases to 5.45of drug-BSA kcal/mol interaction [32,40]. We due also to observed relatively that low the binding drug interactsfree energy with found nearly in − − thefourteen range interaction of −4.35 to residues −5.45 kcal/mol with mainly [32,40]. three We important also observed typesof that interactions the drug suchinteracts as H-bonds, with nearly polar fourteenand hydrophobic interaction interactions. residues with Our mainly model indicatesthree important that the types probable of interactions binding pocket such of as luminespib H-bonds, polarshows and three hydrophobic hydrogen bond interactions. interactions, Our five model polar andindicates six hydrophobic that the interactionsprobable binding with BSA pocket residues of luminespib(as shown inshows Table three2). From hydrogen the docking bond interactions, simulation, five we polar believe and that six hydrophobic the probable interactions binding site with for BSAthe luminespibresidues (as is shown hydrophilic in Table domain 2). From I of BSA,the do Figurecking3 a.simulation, The stable we hydrogen believe that bond the interaction probable bindingbetween site amino for the acid luminespib residues such is hydrophilic as Tyr 147, Ser domain 428 and I of Ser BSA, 192 Figure and luminespib 3a. The stable is shown hydrogen in Figure bond3b. interactionThis may be between due to theamino anionic acid natureresidues of such luminespib as Tyr 147, that Ser helps 428 in and forming Ser 192 hydrogen and luminespib bond interactions. is shown in Figure 3b. This may be due to the anionic nature of luminespib that helps in forming hydrogen bond interactions. In addition, it also has amine-containing heterocyclic functional group to stabilize

Polymers 2020, 12, 1798 8 of 17

Polymers 2020, 12, x FOR PEER REVIEW 8 of 17 In addition, it also has amine-containing heterocyclic functional group to stabilize the protein-ligand the protein-ligand complex via. hydrogen bond interactions as seen with other pharmacophores complex via. hydrogen bond interactions as seen with other pharmacophores [41,42]. [41,42].

FigureFigure 3.3. ((aa)) BindingBinding pocketpocket forfor luminespibluminespib inin BSA;BSA; ((bb)) aminoamino acidacid residuesresidues ofof BSABSA thatthat interactinteract withwith luminespibluminespib throughthrough hydrogen hydrogen bond bond interactions. interactions. It alsoIt also shows shows the the interaction interaction bonding bonding distance distance for thefor hydrogenthe hydrogen bonds bonds when when BSA BSA protein prot interactsein interacts with with luminespib. luminespib.

Table 2. The amino acid residues of BSA (PDI: 3V03) that interacts with luminespib to form a stable Table 2. The amino acid residues of BSA (PDI: 3V03) that interacts with luminespib to form a stable protein-ligand complex. protein-ligand complex.

HydrogenHydrogen Bond Bond Polar Cation-pi Cation-pi Hydrophobic Hydrophobic Other Other TYR147TYR147 ARG458ARG458 TYR451TYR451 ALA193ALA193 LEU189LEU189 ( 0.6974) ( 1.526) ( 0.6994) ( 0.5145) ( 1.6106) −(−0.6974) (−1.526) (−0.6994)− (−−0.5145) (−−1.6106) SER428SER428 HIS145 PRO146PRO146 THR190THR190 ( 0.04736) ( 1.1971) ( 0.4804) ( 0.6233) −(−0.04736) (−1.1971) (−−0.4804) (−−0.6233) SER192SER192 ARG196ARG196 ILE455ILE455 ( 0.4233) ( 1.0906) ( 0.4311) −(−0.4233) (−1.0906) (−−0.4311) GLU424GLU424 ( 0.5991) (−0.5991) ASP108ASP108 ( 0.479) (−0.479)

InIn orderorder toto confirmconfirm thatthat thethe drugdrug interactsinteracts withwith thethe BSA,BSA, wewe performedperformed fluorescencefluorescence quenchingquenching experiment.experiment. Here,Here, wewe kept kept the the concentration concentration of of BSA BSA as as a constanta constant (12.5 (12.5µM) µM) and and stepwise stepwise increased increased the concentrationthe concentration of luminespib of luminespib solution solution (3.125 (3.125µM to 12.5µM µtoM). 12.5 We µM). observed We observed a great drop a great or quenching drop or inquenching the fluorescent in the fluorescent intensity withintensity the with increase the increa in these drugin the concentration drug concentrat asion shown as shown in Figure in Figure4a. This4a. This indicates indicates that that the drugthe drug interacts interacts with with BSA, BSA, and quenchingand quenching is attributed is attributed to the to changes the changes in the in BSA the microenvironmentBSA microenvironment due todue the to drug the drug interaction interaction [43]. [43]. We alsoWe also recorded recorded the fluorescencethe fluorescence spectra spectra for nativefor native BSA BSA NPs NPs and and compared compared it to it thatto that of DNPsof DNPs at aat concentration a concentration of 1of mg 1 mg/mL./mL. It wasIt was observed observed a nearlya nearly 8-fold 8-fold di ffdifferenceerence as shownas shown in Figurein Figure4c. The4c. The decrease decrease in the in relativethe relative fluorescence fluorescence intensity intensity for DNPsfor DNPs can can be attributedbe attributed to theto the formation formation of of drug-BSA drug-BSA complex complex [32 [32].]. The The presence presence of of thethe drug-BSAdrug-BSA complexcomplex waswas alsoalso confirmedconfirmed fromfrom thethe UVUV absorptionabsorption peakspeaks asas shownshown inin FigureFigure4 b.4b. The The drug drug signal signal atat around 310 nm nm in in DNPs DNPs that that coincides coincides with with pure pure drug drug signal signal as positive as positive control control was wasobserved. observed. The signal was absent in native BSA NPs. Overall, UV and fluorescence spectroscopy confirms the

Polymers 2020, 12, 1798 9 of 17

PolymersThe signal 2020, 12 was, x FOR absent PEER in REVIEW native BSA NPs. Overall, UV and fluorescence spectroscopy confirms9 of the17 interaction between ligand and protein. Luminespib probably interacts with BSA through various interaction between ligand and protein. Luminespib probably interacts with BSA through various non-covalent forms of interactions (indicated in Table2) for the formation of self-assembled DNPs. non-covalent forms of interactions (indicated in Table 2) for the formation of self-assembled DNPs.

FigureFigure 4. 4. (a(a) )Fluorescence Fluorescence quenching quenching when when BSA BSA interacts interacts wi withth luminespib luminespib while while formation formation of of drug drug BSABSA nanoconjugates; nanoconjugates; (b ()b DNPs) DNPs when when subjected subjected to UV to UVabsorptions absorptions studies, studies, it shows it shows presence presence of a peak of a aroundpeak around 310 nm, 310 the nm, same the sameas that as thatof the of thenati nativeve drug drug and and blank blank BSA BSA NPs NPs as as controls; controls; (c ()c )DNP DNP nanodispersionnanodispersion in in PBS PBS 7.4 7.4 pH pH at at 1 1mg/mL mg/mL concentr concentrationation was was studied studied with with fluorescence fluorescence spectroscopy spectroscopy wherewhere native native BSA BSA NPs NPs we werere taken taken as as control. control.

3.3.3.3. X-ray X-ray Photo Photo Electron Electron Spectr Spectroscopy,oscopy, TLC TLC and and Stability Stability Studies Studies Further,Further, to to confirm confirm the the presence presence of of the the drug drug in interactionteraction with with BSA BSA we we pe performedrformed XPS XPS analysis. analysis. It It cancan be be seen seen from Figure1 1aa that that the the drug drug consists consists of of nitrogen- nitrogen- and and oxygen- oxygen- containing containing heterocyclic heterocyclic ring ringstructure. structure. Hence, Hence, the presencethe presence of these of these ring systemsring systems in DNP in conjugatesDNP conjugates may provide may provide relative relative shifts or shiftschanges or changes in the atomic in the signalsatomic forsignals carbon for andcarbon nitrogen and nitrogen due to the due electron-withdrawing to the electron-withdrawing and donating and donatingmicroenvironment microenvironment of DNPs inof comparisonDNPs in comparis to nativeon BSA to NPs.native Figure BSA 5NPs.a shows Figure the comparison5a shows the of comparisoncarbon signatures of carbon from signatures DNPs, native from BSADNPs, NPs nati andve drug.BSA NPs It was and observed drug. It thatwas –N–Cobserved=O bond that –N– peak C=Owas observedbond peak at was 286.45 observed for BSA, at which 286.45 was for shiftedBSA, which to 287 was (shifted shifted by 1.05to 287 eV) (shifted for DNPs. by This1.05 eV) positive for DNPs.shift may This be positive attributed shift to may the be presence attributed of electron to the presence withdrawing of electron aromatic withdrawing nucleus of aromatic the drug nucleus present ofin the DNPs. drug Further, present we in alsoDNPs. observed Further, three-peak we also signaturesobserved three-peak for DNPs andsignatures drug sample for DNPs that wasand absentdrug samplein native that BSA was NPs absent as shown in native in Figure BSA 5NPsa. In as this shown three-peak in Figure signature, 5a. In this the peakthree-peak at 285 signature, eV (Figure the5a) peakis correspondent at 285 eV (Figure to –C 5a)=N is bond, correspondent which corresponds to –C=N tobond, the –Cwhich=N– corresponds of iso-oxazole to moietythe –C=N– of the of drugiso- oxazolemolecule moiety (Figure of1 thea) [44drug]. molecule (Figure 1a) [44]. Moreover, to understand the nature of changes in carbon signals, we analyzed the relative changes in the nitrogen signatures. It was observed that XPS peak for =N–O– was absent in native BSA, but it was present in drug and DNP spectra as shown in Figure 5b. The =N–O– peak was found to be slightly shifted to 401.3 for DNPs than native drug where it was found to be at 399.75 eV [45]. These results also suggest the drug-protein interactions, which is consistent with the fluorescence and UV.

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FigureFigure 5. 5. XPSXPS spectra spectra for for carbon carbon 1s 1s (C (C 1s) 1s) and and nitrogen nitrogen 1s 1s (N (N 1s) 1s) alterations alterations in in binding binding energy energy peaks peaks forfor DNPs DNPs in in comparison comparison to to native native BSA BSA NP NP and and luminespib luminespib as as control. control. ( (aa)) C C 1s 1s shifts shifts also also mark mark the the presencepresence of of peak peak at at second second position position for for drug drug and and DNPs DNPs whereas whereas its its absence absence in in native native BSA BSA NPs; NPs; ( (bb))N N 1s1s peak shiftsshifts andand the the presence presence of of peak peak at positionat position one one for drugfor drug and and DNPs DNPs whereas whereas its absence its absence in native in nativeBSA NPs. BSA PeakNPs. Peak fitting fitting function functi byon multipack by multipack software, software, helps helps in identification in identification of multipleof multiple peaks peaks in inthe the raw raw data. data. The The sharp sharp solid solid lines lines in in above above spectra spectra represents represents peak peak fits fits for for identification identification of of single single or ormultiple multiple peaks peaks in in the the wavy wavy lines lines of of raw raw data. data.

Furthermore,Moreover, to understandto confirm thethe nature presence of changes of drug in carbonin DNP signals, nanoconjugates, we analyzed we the performed relative changes TLC studiesin the nitrogen in comparison signatures. to native It was drug observed and BSA that NPs XPS as peak shown for =inN–O– Figure was 6. It absent was observed in native BSA,that 10 but µL it spotwas presentof DNPs in at drug 1 mg/mL and DNP concentration spectra as shown in deionized in Figure water5b. The provided=N–O– an peak average was found Rf value to be of slightly 0.64 ± 0.03.shifted However, to 401.3 ethanol for DNPs solution than native of native drug drug where gave it wasRf value found ofto 0.85 be ± at 0.005 399.75 (as eV shown [45]. Thesein Figure results 6). Thesealso suggest observations the drug-protein clearly show interactions, the presence which of isdr consistentug in DNPs with in the comparison fluorescence to andpositive UV. (native drug)Furthermore, and negative to (BSA confirm NPs) the control presence [31]. of The drug difference in DNP nanoconjugates, in the Rf values we for performed the native TLC drug studies and DNPsin comparison is due to tothe native interaction drug of and drug BSA with NPs BSA as shownin DNP in formulation. Figure6. It wasThis observedresult also that supported 10 µL spotour previousof DNPs experimental at 1 mg/mL concentration results provided in deionized by fluorescence water providedand UV spectroscopy. an average R value of 0.64 0.03. f ± However, ethanol solution of native drug gave R value of 0.85 0.005 (as shown in Figure6). f ± These observations clearly show the presence of drug in DNPs in comparison to positive (native drug) and negative (BSA NPs) control [31]. The difference in the Rf values for the native drug and DNPs is due to the interaction of drug with BSA in DNP formulation. This result also supported our previous experimental results provided by fluorescence and UV spectroscopy.

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µ FigureFigureFigure 6.6. 6. Thin-layerThin-layer Thin-layer chromatographychromatography chromatography (TLC)(TLC (TLC) ) imageimage image withwith with thethe the 1010 10 µL LµL spotspot spot ofof of DNPsDNPs DNPs inin in comparisoncomparison comparison toto to native BSA NPs and luminespib on TLC plate as controls. The solvent front was run for 144 cm to nativenative BSA BSA NPs NPs and and luminespib luminespib on on TLC TLC plate plate as as controls. controls. The The solvent solvent front front was was run run for for 144 144 cm cm to to calculate Rf values for respective mobility of spots for BSA NPs, DNPs and native drug. Our results calculatecalculate R Rf fvalues values for for respective respective mobility mobility of of spots spots for for BSA BSA NPs, NPs, DNPs DNPs and and native native drug. drug. Our Our results results show a real time shifts in the spot position for luminespib from DNPs in comparison to native drug. showshow a a real real time time shifts shifts in in the the spot spot position position for for luminespib luminespib from from DNPs DNPs in in comparison comparison to to native native drug. drug. The calculated Rf values were performed n = 3 times and average were taken to determine the shift in TheThe calculated calculated R Rf fvalues values were were performed performed n n = = 3 3 times times and and aver averageage were were taken taken to to determine determine the the shift shift in in the position of spots. thethe position position of of spots. spots. It was also important to check the stability of the formulation to understand the storage condition ItIt waswas alsoalso importantimportant toto checkcheck thethe stabilitystability ofof thethe formulationformulation toto understandunderstand thethe storagestorage of the formulation. Most nanoparticles preparations can be stored under room temperature [46,47]. conditioncondition of of the the formulation. formulation. Most Most nanoparticles nanoparticles preparations preparations can can be be stored stored under under room room temperature temperature To assess this, we performed stability evaluation for our preparation. As shown in Figure7a,b. There [46,47].[46,47]. To To assess assess this, this, we we performed performed stability stability eval evaluationuation for for our our preparation. preparation. As As shown shown in in Figure Figure was insignificant increase in the particle size from 220 nm (Day 0) to 255 nm (Day 7) and polydispersity 7a,b.7a,b. There There was was insignificant insignificant increase increase in in the the particle particle size size from from 220 220 nm nm (Day (Day 0) 0) to to 255 255 nm nm (Day (Day 7) 7) and and index from 0.133 (Day 0) to 0.176 (Day 7). Further, we also observed that there was relative increase polydispersitypolydispersity index index from from 0.133 0.133 (Day (Day 0) 0) to to 0.176 0.176 (D (Dayay 7). 7). Further, Further, we we also also observed observed that that there there was was in number of particles in the size range of around 500 to 700 nm. This could be due to the polymer relativerelative increase increase in in number number of of particles particles in in the the size size range range of of around around 500 500 to to 700 700 nm. nm. This This could could be be due due swelling over time or due to particle aggregations. To check the visual observation, SEM was performed. toto the the polymer polymer swelling swelling over over time time or or due due to to partic particlele aggregations. aggregations. To To check check the the visual visual observation, observation, Overall, data from particle size distribution and SEM suggests that the nanoparticles are stable at room SEMSEM waswas performed.performed. Overall,Overall, datadata fromfrom particleparticle sizesize distributiondistribution andand SEMSEM suggestssuggests thatthat thethe temperature for nearly 7 days, but may aggregated over time at 25 C. Hence, dispersions require nanoparticlesnanoparticles are are stable stable at at room room temperature temperature for for nearly nearly 7 7 days, days, but but may◦ may aggregated aggregated over over time time at at 25 25 necessary sonication before use. °C.°C. Hence, Hence, dispersions dispersions require require necessary necessary sonication sonication before before use. use.

Figure 7. (a) Particle size distribution and (b) SEM image of the nanoparticles stored at 25 ◦C for 7 days. FigureFigure 7. 7. ( a(a) )Particle Particle size size distribution distribution and and ( b(b) )SEM SEM image image of of the the nanoparticles nanoparticles stored stored at at 25 25 °C °C for for 7 7 days. days. 3.4. In Vitro Drug Release Studies 3.4.3.4. In In Vitro Vitro Drug Drug Release Release Studies Studies The drug release studies of DNP formulation were performed under pH 7.4 for 72 h. It was observedTheThe that drugdrug the releaserelease first burst studiesstudies release ofof DNPDNP of the formulationformulation drug occurred werewere in performedperformed the first 8 underhunder of incubation pHpH 7.47.4 forfor period. 7272 h.h. ItIt can waswas observedobserved that that the the first first burst burst release release of of the the drug drug occu occurredrred in in the the first first 8 8 h h of of incubation incubation period. period. It It can can bebe seen seen that that there there was was a a time-dependent time-dependent exponential exponential increase increase in in the the drug drug release release for for 24 24 h. h. This This was was

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Polymers 2020, 12, x FOR PEER REVIEW 12 of 17 be seen that there was a time-dependent exponential increase in the drug release for 24 h. This was followedfollowed by by sustained sustained release release of of drug drug from from DNP DNP as as shown shown in in Figure Figure8. 8. This This study study clearly clearly shows shows that that wewe may may expect expect to to see see the the desired desired pharmacological pharmacological activity activity within within 24 24 to to 48 48 h. h.

FigureFigure 8. 8.Time-dependent Time-dependent sustained sustained release release of of drug drug from from DNPs DNPs over over a a period period of of 72 72 h. h. 3.5. In Vitro Cytotoxicity Studies 3.5. In Vitro Cytotoxicity Studies To evaluate the anticancer therapeutic efficacy of synthesized DNPs, cytotoxicity studies were To evaluate the anticancer therapeutic efficacy of synthesized DNPs, cytotoxicity studies were performed on cancer cell lines such as MIA PaCa-2 and MCF-7. We also checked the effect of DNPs on performed on cancer cell lines such as MIA PaCa-2 and MCF-7. We also checked the effect of DNPs normal mice fibroblast (L929) cell line. Both, BSA NP treatment for MCF-7, MIA PaCA-2, L929 and on normal mice fibroblast (L929) cell line. Both, BSA NP treatment for MCF-7, MIA PaCA-2, L929 and DNP against L929 were taken as ve controls for the analysis. In addition, native drug treated MCF-7, DNP against L929 were taken as− −ve controls for the analysis. In addition, native drug treated MCF- MIA PaCa-2 and L929 were taken as +ve control for the cytotoxicity assay (Figure 10). The cytotoxicity 7, MIA PaCa-2 and L929 were taken as +ve control for the cytotoxicity assay (Figure 10). The for all the test and ve control group was recorded at 24 and 48 h intervals as shown in Figure9. cytotoxicity for all− the test and −ve control group was recorded at 24 and 48 h intervals as shown in We were able to see a concentration dependent cytotoxicity for DNPs towards MIA PaCa-2 and Figure 9. MCF-7 cell line in comparison to L929 as shown in Figure9a,b (with p < 0.05). Moreover, a similarity We were able to see a concentration dependent cytotoxicity for DNPs towards MIA PaCa-2 and in the therapeutic efficacy of DNPs formulation towards MIA PaCa-2 and MCF-7 cell lines for all MCF-7 cell line in comparison to L929 as shown in Figure 9a,b (with p < 0.05). Moreover, a similarity the concentrations (0.1 to 1 mg/mL) was also observed, as shown in Figure9a,b. At, 24 and 48 h of in the therapeutic efficacy of DNPs formulation towards MIA PaCa-2 and MCF-7 cell lines for all the incubation for all the three cell lines, DNPs exhibited relatively high cytotoxicity towards cancer cell concentrations (0.1 to 1 mg/mL) was also observed, as shown in Figure 9a,b. At, 24 and 48 h of lines (MIA PaCa-2 and MCF-7) compared to normal L929 cell line. incubation for all the three cell lines, DNPs exhibited relatively high cytotoxicity towards cancer cell Moreover, it was also observed that for MIA PaCa-2 and MCF-7 cell lines, DNPs shows good cell lines (MIA PaCa-2 and MCF-7) compared to normal L929 cell line. growth inhibition at 1 mg/mL concentration in 24 h and 48 h intervals (p < 0.05) as shown in Figure9. Moreover, it was also observed that for MIA PaCa-2 and MCF-7 cell lines, DNPs shows good After 24 h of incubation we observed that DNPs at 1 mg/mL concentration, the percentage of cell cell growth inhibition at 1 mg/mL concentration in 24 h and 48 h intervals (p < 0.05) as shown in viability for MIA PaCa-2 and MCF-7 cells were found to be around 62% and 63%, respectively. After 48 h, Figure 9. After 24 h of incubation we observed that DNPs at 1 mg/mL concentration, the percentage the viability further dropped to around 53% and 52% for MIA PaCa-2 and MCF-7, respectively. A similar of cell viability for MIA PaCa-2 and MCF-7 cells were found to be around 62% and 63%, respectively. variation in percentage of cell viability of MIA PaCa-2 and MCF-7 was seen for other concentrations of After 48 h, the viability further dropped to around 53% and 52% for MIA PaCa-2 and MCF-7, DNPs (with p < 0.05) as shown in Figure9. Our results clearly shows selective cancer killing by DNPs respectively. A similar variation in percentage of cell viability of MIA PaCa-2 and MCF-7 was seen due to the presence of Hsp90 inhibitor (luminespib) in them [22]. In comparison to standard free drug for other concentrations of DNPs (with p < 0.05) as shown in Figure 9. Our results clearly shows (luminespib), DNPs formulations were shown to have controlled and sustained cytotoxic (over 24 and selective cancer killing by DNPs due to the presence of Hsp90 inhibitor (luminespib) in them [22]. In 48 h) effect in comparison to free drug (Figure 10) for all the three concentrations against all the three comparison to standard free drug (luminespib), DNPs formulations were shown to have controlled cell lines. and sustained cytotoxic (over 24 and 48 h) effect in comparison to free drug (Figure 10) for all the three concentrations against all the three cell lines.

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Figure 9.9. ((a,,b)) PercentPercent cellcell viabilityviability ofof MIA MIA PaCa-2 PaCa-2 and and MCF-7 MCF-7 (cancer) (cancer) cell cell lines lines treated treated with with DNPs DNPs in comparisonin comparison to to treatment treatment of of L929 L929 as as control. control. Figure Figure shows shows comparative comparative account account forfor cellcell lineslines treatedtreated withwith blankblank BSABSA NPsNPs andand DNPs.DNPs. It also shows the cytotoxic and biocompatible eeffectffect exhibitedexhibited by DNPs and nativenative BSABSA NPs,NPs, respectively,respectively, inin bothboth concentrationconcentration andand aa time-dependenttime-dependent manner.manner. We observed thatthat blank blank BSA BSA NPs NPs and DNPsand DNPs are biocompatible are biocompatible towards towards L929 in aL929 concentration- in a concentration- dependent mannerdependent compared manner tocompared that of MIA to that PaCa-2 of MIA and PaCa-2 MCF-7 and with MCF-7 * p < with0.05. * p The < 0.05. comparison The comparison was drawn was betweendrawn between groups (betweengroups (between test and controltest and groups control as wellgroups as betweenas well twoas between time points) two fortime calculation points) for of statisticalcalculation significance. of statistical significance.

Our results aboutabout thethe biocompatibilitybiocompatibility of BSA encapsulatedencapsulated drugs formulation and bare BSA NPs against MIA PaCa-2, MCF-7 and L929 align with previously reported studies about the BSA conjugated nanoformulations [[25,32,48]25,32,48] as shown inin FiguresFigures1 1bb and and9 .9. Hence, Hence, aqueous aqueous dispensable dispensable luminespib-loadedluminespib-loaded BSA BSA NPs NPs may may prove prove to beto be an importantan important step step towards towards the use the of use this of formulation this formulation for its anticancerfor its anticancer effect due effect to Hsp90 due inhibitionto Hsp90 againstinhibition breast against cancer breast and pancreatic cancer and cancer. pancreatic The representative cancer. The Figurerepresentative1c and cytotoxicity Figure 1c and assay cytotoxicity in Figure9a,b assay clearly in Fi showgure anticancer9a,b clearly behavior show an ofticancer our DNP behavior formulation of our againstDNP formulation cancer cell against lines, which cancer may cell lines, be attributed which may to drug be attributed release over to drug a period release of over time a (shown period inof Figuretime (shown8). These in Figure results 8). clearly These show results that clearly our aqueous show that dispensable our aqueous DNP di formulationspensable DNP may formulation prove to be amay viable prove option to be for a viable breast option cancer andfor breast pancreatic cancer cancer and pancreatic therapy. cancer therapy.

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FigureFigure 10. (a10.–c )( Percenta–c) Percent relative relative cytotoxicity cytotoxicity of native of native luminespib luminespib in concentration in concentration and time-dependentand time- mannerdependent towards manner MiaPaCa-2, towards MCF-7 MiaPaCa-2, and L929 MCF-7 cell lines.and L929 The studycell lines. shows The statistical study shows significance statistical of *, **,

*** p significance< 0.05 within of *, various **, *** p < groups 0.05 within marked various in figure.groups Themarked student in figure.t test The carried student out t test by carried comparing out the lowerby concentration comparing the (100 lowerµg concentration/mL) treated (100 group µg/mL) with treated high concentration group with high (500 concentration and 1000 µ g(500/mL) and treated 1000 µg/mL) treated group for each cell line. group for each cell line.

4. Conclusions4. Conclusions We have developed luminespib-loaded BSA NPs (DNPs) for breast and pancreatic cancer We have developed luminespib-loaded BSA NPs (DNPs) for breast and pancreatic cancer therapy therapy that can be dispensed in an aqueous vehicle. Our fluorescence quenching experiments show that can be dispensed in an aqueous vehicle. Our fluorescence quenching experiments show that that luminespib interacts with BSA and forms nanoparticles by classical desolvation method. Our luminespibdocking interacts study predicted with BSA that andthe probable forms nanoparticles binding site of by the classical luminespib desolvation in BSA during method. formation Our docking of studyDNPs predicted with lowest that theGibbs probable free energy binding of binding site ofof thedrug-BSA luminespib complex in is BSA around during hydrophilic formation domain of DNPs withI lowest of BSA. Gibbs Our cellular free energy biocompatibility of binding studies of drug-BSA also demonstrate complex isthe around safety profile hydrophilic in a concentration- domain I of BSA. Our cellulardependent biocompatibility manner for DNPs studies and native also demonstrateBSA NPs in normal the safety L929 profile cell line in in a concentration-dependentcomparison to cancer mannercell forlines DNPs such as and MIA native PaCa-2 BSA and NPs MCF-7. in normal We also L929 observed cell line significant in comparison (p < 0.05) toanticancer cancer celleffect lines for such as MIAour PaCa-2DNPs in and comparison MCF-7. to We native also observedBSA NPs. We significant believe that (p < this0.05) difference anticancer is due eff ectto non-covalent for our DNPs in comparisonconjugation to native of drug BSA in DNPs NPs. in We comparison believe thatto native this diBSAfference NPs. We is found due to that non-covalent the anticancer conjugation efficacy of of our DNP formulation was similar for MIA PaCa-2 and MCF-7 cell lines. We believe that this work drug in DNPs in comparison to native BSA NPs. We found that the anticancer efficacy of our DNP can be the first step in the direction of improving the formulation Hsp90 inhibitors by using natural formulationbiomaterials was like similar BSA. for MIA PaCa-2 and MCF-7 cell lines. We believe that this work can be the first step in the direction of improving the formulation Hsp90 inhibitors by using natural biomaterials like BSA.Author Contributions: Conceptualization, A.K.R. and D.S.K.; methodology, A.K.R., S.B. and A.R.G.; data collection and analysis, A.K.R., S.B. and A.R.G.; manuscript writing, A.K.R.; review and editing, S.B., A.R.G., AuthorD.S.K. Contributions: and G.K.; supervision,Conceptualization, T.M., G.K. and D.S.K.; A.K.R. proj andect D.S.K.;administration, methodology, T.M. and A.K.R.,D.S.K.; funding S.B. and acquisition, A.R.G.; data collectionA.K.R., and G.K., analysis, and D.S.K. A.K.R., All authors S.B. and have A.R.G.; read and manuscript agreed to the writing, published A.K.R.; version review of the manuscript. and editing, S.B., A.R.G., D.S.K. and G.K.; supervision, T.M., G.K. and D.S.K.; project administration, T.M. and D.S.K.; funding acquisition, A.K.R.,Funding: G.K., and This D.S.K. research All was authors funded have by Ministry read and of agreedEducation to Culture, the published Sports, versionScience and of the Technology manuscript. (MEXT), Japan for providing Monbukagakusho fellowship, Inoue Enryo Memorial Research Grant, Toyo University, and Funding:programThis of research the strategic was research funded foundation by Ministry for of priv Educationate universities-S1101017, Culture, Sports, by Science MEXT andJapan. Technology The APC was (MEXT), Japanfunded for providing by Thomas Monbukagakusho Jefferson University fellowship, Open Access Inoue fund. Enryo Memorial Research Grant, Toyo University, and program of the strategic research foundation for private universities-S1101017, by MEXT Japan. The APC was funded by Thomas Jefferson University Open Access fund.

Acknowledgments: Authors would also like to thank Pam Walter, Office for Professional Writing, Publishing, & Communication, Thomas Jefferson University for carefully reading and editing this manuscript. Conflicts of Interest: The authors declare no conflict of interest. Polymers 2020, 12, 1798 15 of 17

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