Immunomodulatory and Antitumor Activity of Surface-Functionalized Lactoferrin Nanoparticles in Ovarian Carcinoma

Immunomodulatory and Antitumor Activity of Surface-Functionalized Lactoferrin Nanoparticles in Ovarian Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Immunomodulatory and Antitumor Activity of Surface-Functionalized Lactoferrin Nanoparticles in Ovarian Carcinoma Drishti Panjwani, Asha Patel, Shruti Patel, Nishabh Kushwaha, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8494829/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 19 You are reading this latest preprint version Abstract Objective The study aimed fabricate Anti-Her2 carfilzomib loaded lactoferrin nanoparticles in order to address the shortcomings of carfilzomib related to poor half life, low solubility, and poor in vivo stability. The objective was to modify the programmable assembly of protein with biomarkers and imaging probe to ameliorate precise targeted theragnostics against ovarian cancer Method Carfilzomib loaded lactoferrin nanoparticles were fabricated using Desolvation technique to obtain optimal composition using Full factorial design. Further, surface functionalization with FITC tagged anti-Her2(Ab) was performed using EDC-NHS carbodiimide chemistry. Detailed characterization and in vitro evaluation including cellular uptake studies in SKOV-3 cells were performed. Results The carfilzomib loaded lactoferrin nanoparticles(CFZ-LF-NPs) were found to be uniformly distributed in range of 100-150nm. The percentage drug loading and entrapment efficiency was found to be 14.14 ± 0.521% and 86% ± 0.289, respectively. CFZ-LF-NPs showed prolong drug release till 8 days at physiological pH(7.4), while 91.932 ± 0.549% of drug release at tumor pH 5.5 on day 4, thereby indicating pH triggered drug release. NMR, and fluorescent microscopy confirms successful conjugation of anti-Her2(Ab) with CFZ-LF-NPs. ELISA test for anti-HER2 conjugated NPs(FITC-AntiHer2-CFZ-LF-NPs) revealed the highest percent of antibody(> 60 ± 0.225%) conjugation with nanoparticles above the concentration of 5 ug/ml.The MTT assay, cellular uptake assay and tumor growth inhibition studies in treated mice unravels high cytotoxicity in nanoconjugates(FITC-AntiHer2-CFZ-LF-NPs) as compared to non-conjugated NPs and plain drug. Conclusion The potential approach may emerge in future as a molecular-docks comprising a immunotherapy-based theragnostic in cancer. Biofunctionalization Lactoferrin self-assembly Anti-Her2 Cellular imaging Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 1. Introduction Ovarian cancer(OC) is one of the most common gynaecological cancer with high morbidity, and prevailing cause of female community leading cause of death worldwide[ 1 – 4 ]. This is majorly because of its asymptomatic nature that leads to advanced stage diagnosis and thereby resulting in malignant ascites with poor prognosis[ 5 , 6 ]. According to GLOBOCAN, the incidence rates for OC increased to 315,949 with 300000 deaths worldwide[ 7 ]. Despite of numerous efforts that have been made using traditional treatments including chemotherapy, in which surmounting undesired off-target effects, recurrence, immune-suppression and emergence of multi-drug resistance remains a challenge[ 8 ]. Therefore, this have created an urge for employing precise and selective targeting of heterogenous tumor markers, immune modulation, and targeting proteins that are deranged in ovarian cancer. Proteomics is a promising approach in targeting the proteins that are deranged in cancer[ 9 , 10 ]. Hence, precise dysregulation of Ubiquitin proteosome pathway can be a potential target in cancer, wherein terminating the protein degradation leading to ER stress followed by cyclin pathway mediated cell death[ 11 ]. Carfilzomib is a front line proteosome inhibitor, with high potency to bind irreversibly at active site of immune proteosome and inhibit immune checkpoints. Despite of potential clinical outcomes, it is associated [ 12 – 14 ] with some limitations including rapid clearance(< 30mins), poor biostability results in incomplete dose maintenance at tumor site, and low solubility[ 15 ]. To mitigate these shortcomings, combining therapeutics of such immune checkpoint inhibitors like carfilzomib with functional nanomaterial that can enhance the targeting affinity, and drug retention at target site[ 16 – 22 ]. Proteins like lactoferrin have garnered superior attention as nanocarriers due to its multi-functional role as anti-inflammatory, anti-microbial, immune modulatory, and ability to employ iron homeostasis[ 23 – 25 ]. Lactoferrin is iron containing glycoprotein of transferrin family with potential ability to scavenge free iron at inflamed sites, that enables iron deficiency in cancer cells, thereby contributing to cell death[ 26 ]. In contrast to synthetically derived polymers releasing toxic metabolites upon degradation, lactoferrin based proteins as natural building blocks get metabolized into amino acids which can be used by the body tissues. Additionally, proteins and iron remain in high demand by proliferating cancer cells, thereby overexpresses transferrin receptor that favor receptor mediated uptake of cargoes containing lactoferrin nanocarriers[ 27 ]. These multifunctional role makes lactoferrin an ideal drug carrier that not only carries the drug to target site but also synergistically contributes with drug in therapeutically deceiving the cancer cells. However, proteomics delivery endures interaction with serum proteins, thereby increasing a surge for progressive advancement in genetic and molecular modalities in development of target specific biomarker-driven therapy[ 9 , 28 – 31 ]. In this context, we developed Anti-Her2 tagged carfilzomib loaded lactoferrin nanoparticles as Her2 specific functional nanomaterials for immune-biotherapeutics, designed to integrate immune-checkpoint inhibition with antibody-mediated tumor targeting in ovarian cancer. Moreover, lactoferrin is capable of targeting transferrin receptor overexpressed on cancer cells requiring iron for proliferation. However, concerning with tumor heterogenicity in OC phenotypes, modifying the programmable assembly of lactoferrin by altering the surface with active ligand molecules for active targeting facilitates dual targeting and combined therapeutics. Based on reports, ameliorating multifunctional targeting by employing molecular tagging on proteins like lactoferrin that can not only carry the drug to target site but also synergistically contribute in enhancing apoptosis in cancer cells[ 32 ]. In this work, AntiHer2 was conjugated with nanoparticles using carbodiimide chemistry[ 3 ], that was confirmed using NMR and ELISA followed by detailed evaluation of anti-tumor effect in SKOV-3 cells. The findings of our research suggests that our developed formulation may emerge as a molecular docks comprising nanomedicine with significant application in biosensing,bio imaging as well as immunotherapy-based theragnostic. 2. Materials and Methods 2.1 Chemicals: Carfilzomib (CFZ) (99.8% pure) was obtained as a gift sample from BioconLtd. Lactoferrin(pharmaceutical grade) and Anti-Her2 was procured from sigma Aldrich, Mumbai. Desolvating solvents like methanol, doubled distilled water, and acetone were procured from chem dyes, India. Other chemicals utilized from chemical synthesis of nanoconjugates of carfilzomib like 1-Ethyl-3-(3 dimethylaminopropyl)carbodiimide(EDC), N-hydroxysuccinimide(NHS) N,N-Dicyclohexylcarbodiimide Glutaraldehyde, were supplied by Sigma Aldrich, Mumbai. HPLC grade solvents were procured from Qualikems, India. SKOV-3 cells were procured from ATCC, USA. Other materials used for cancer cell culture studies such as bioimaging molecular tag(FITC), 4',6-diamidino-2-phenylindole (DAPI), Annexin-V kit, Propidium iodide, Fetal bovine serum(FBS), and Dulbecco’s Modified Eagle Medium(DMEM) were procured from Sigma Aldrich Mumbai. ELISA kit, Cell counting Kit(CCK), cell culture well plates, and trypsin were obtained from Thermofischer Scientific(Shanghai China). 2.2 Synthesis of lactoferrin nanoparticles. Carfilzomib loaded lactoferrin nanoparticles(CFZ-LF-NPs) were fabricated using desolvation technique, which is based on the principle of self-assembly. Firstly, the LF was dissolved in double distilled water to prepare the aqueous solution, and then the prepared solution was filtered through the 0.45µm pore size of Millipore membrane. Further, the pH of aq solution was adjusted to 5 using 0.1N HCl. Further, the drug carfilzomib was dissolved in binary mixture of desolvating agent solution(ethanol:water) in a ratio of 1:1 to 3:1%w/v. The desolvating solution is then added gradually dropwise in aq solution of lactoferrin. The principle of self-assembly is based on protein folding/unfolding transition via disturbing the native environment(pH, temperature, and charge), that enables the exposure of hydrophobic domains for ameliorating [ 33 – 35 ] physical encapsulation of drug. The resultant nanoparticles were further chemically crosslinked by adding varied concentration of glutaraldehyde (0.05–0.1%) into the turbid suspension to rigidize lactoferrin nanoparticles by stirring the nanoparticles to prepare crosslinked carfilzomib loaded lactoferrin nanoparticles. The resulting mixture was subjected for centrifugation ate 5000rpm to eliminate the un-entrapped drug. The resulting supernatant was extracted and the pellets were repeatedly washed with deionized water in order to obtain ‘Carfilzomib loaded lactoferrin nanoparticles’(CFZ-LF-NPs). The nanoparticles were further freeze-dried using Epsilon alpha 1–4 LSC(Martin Christ, Germany) according to the developed and optimized recipe for lyophilization 2.3 Fabrication of Anti-Her2 conjugated lactoferrin nanoparticles. The surface modification of CFZ-LF-NPs was carried out using EDCNHS carbodiimide chemistry. Briefly, 4 mg EDC(0.4%w/v) was dissolved in 1 ml of PBS(0.1M, pH 5.8) containing 1 mg NHS, followed addition of 10µl of anti-Her2(1mg/ml in 0.1M PBS, pH 7.4) forming a suspension under continuous stirring till 30 mins. Further, CFZ-LF-NPs(10mg) were reconstituted in 1 ml of PBS (0.1M pH5.8) and were added in above activated anti-Her2 solution. The resulting solution was allowed to stir continuously for 4 hours followed by incubation at freezing temperature(4⁰C) for 1 hour. Then, resulting was further centrifuged at 10,000 rpm, 4⁰C in a triplicate manner to remove excess EDC and unconjugated residues [ 36 – 38 ]. The nanoconjugates(Ab CFZ-LF-NPs) were repeatedly washed with Milli Q water followed by lyophilization using the optimized method. The nanoconjugates were collected after lyophilization and were subjected for ELISA examination to determine the percent of anti-Her2 conjugation and to measure the amount conjugated with CFZ-LF-NPs. 2.4 FITC conjugation: FITC is prompt to bind with amino acids of antibodies, peptides, and proteins[ 39 , 40 ]. Briefly, the CFZ-LF-NPs and AntiHer2-CFZ-LF-NPs in a molar ratio of 1:2 w/w, were dissolved in sodium carbonate/bi carbonate buffer solution (Na2CO3/NaHCO3, 25mM, pH 9.8) with substantial addition of FITC(0.1mg/ml). The resulting solution was allowed to stir with continuous agitation in incubator shaker at room temperature for 4 hours. The resulting fabricated nanoconjugates were centrifuged at 10,000 rpm, 4⁰C in a triplicate manner to remove excess unconjugated residues. The product was washed repeatedly to obtain FITC-CFZ-LF-NPs and FITC-AntiHer2-CFZ-LF-NPs. 2.5 Nanoparticles characterization and evaluation: Particle size distribution, polydispersity index (PDI) and zeta [ 34 , 41 ]potential of CFZ-LF-NPs and Anti-Her2-CFZ-LF-NPs. was evaluated using dynamic light scattering(DLS, Malvern Z90 zetasizer). Briefly, dispersion of nanoparticles(50mg) was prepared by reconstituting in water for injection(WFI) to prepare 5mg/ml concentration, further dispersing it in water for injection (WFI) to prepare a 5mg/ml, with simultaneous vortexing for 2 minutes at 2.5 vortex level in order to obtain a uniform protein nanoparticulate dispersion. The resulting dispersion of nanoparticles with 1.334 refractive index, was taken in disposable polystyrene cell cuvette and analysed for measurement run at 25ºC and scattering at 90º angle. The hydrodynamic diameter of intensity resulted was discerned by z-avg value. Each experimental run was performed in triplicate and analysed at 25°C for demonstrating distribution pattern of nano particulates, and results were cumulated using the average of triplicate values of samples. Both CFZ-LF-NPs and Anti-Her2-CFZ-LF-NPs were subjected to 1HNMR analysis, which was recorded on Variant 400-MHZ spectrometer for confirming the successful configuration of CFZ-LF-NPs and conjugation of Anti-Her2(AB) with CFZ-LF-NPs. The samples containing freeze dried NPs and nanoconjugates were prepared in a Teflon-sleeved NMR tube(4.0mm o.d., 3.2 mm i.d.,Wilmad Glass) by concurrently dissolving protein-based nanostructures(5mg) in D2O(0.4 ml, 0.6%v/v). The resulting NMR sampling and characterization was handled at ambient temperature and pressure. NMR experiments were performed on 400 and 500 MHz using Tetramethylsilane(TMS) in 1HNMR for chemical shift reference[ 34 , 35 ]. Transmission emission microscopy(TEM) was used for surface morphological examination of lactoferrin nanoconjugates. TEM examination was performed using CM120 microscope. Subsequent dilutions of nanosuspension were prepared in double distilled water and a small quantity of suspension was deposited on a grid coated with polymer-based copper film. It was then allowed to settled for few mins.Excess liquid was scrapped off, and uranyl acetate was added to the sample followed by allowing it to dry at room temperature to form a thin film which was further examined at 70kv[ 35 , 42 ]. Further, The FITC concentration post conjugation with Ab-CFZ-LF-NP was determined using fluorescence spectrophotometer. Fluorescence spectra of FITC labelled nanoconjugates were obtained on a Jasco spectrofluorometer using a 1cm path length cell containing the nanoconjugates at concentration ranging 5–20 µg at room temperature(25⁰C)[ 39 , 40 ]. The samples were prepared by diluting in PBS solution and the spectra were examined at excitation wavelength of 495nm. The emission spectra were recorded between 200–800 nm. The %EE of carfilzomib was evaluated and determined by HPLC-based developed method. The Carfilzomib peaks were integrated at 210nm. For determination of %EE of CFZ-LF-NPs and Anti-Her2-CFZ-LF-NPs, the nanoparticles were weighed at equal concentration of 2mg/ml and centrifuged at 10,000 rpm for two cycles by repeatedly dispersing the pellets in acetonitrile in order to separate the unentrapped drug and further collected supernatant was analysed for entrapment efficiency after computing the chromatograms resulted by validated HPLC analysis[ 43 , 44 ]. Drug release from the synthesized nanoparticles was performed using the dialysis bag method (cut-off 12–14 kDa). The %DR was evaluated at both physiological condition (pH 7.4) and the conditions prevalent in tumor (pH 5.5). Drug release was quantified by measuring the concentration of the released drug(CFZ) in the surrounding solution of at regular time intervals using a validated HPLC method of analysis at 210nm. The stability of the optimal formulation in suspension was evaluated for 6 months at at 4–8°C ± 2°/45%±5% RH ( Refrigerator, RF) and 25°C ± 2°C/65 ± 5% RH ( Room temperature; RT) as per ICH guidelines. Samples were evaluated, at 15-30days interval. The physiochemical change in protein nanoparticles was recorded periodically by evaluating %EE, size distribution, and physical appearance[ 21 ]. 2.6 ELISA test for examining anti-Her2 concentration in nanoconjugates (FITC-Anti-Her2-CFZ-LF-NPs): The amount of anti-Her2 conjugated with CFZ-LF-NPs was determined using ELISA plate reader(BioRad, USA). The sample aliquots ranging 100-600ng/ml of Ab-CFZ-LF-NPs, were prepared in 0.1M PBS (pH7.4) and ELISA plate reading was employed using the developed protocol as follows; The plates were washed thrice after incubation with PBS (0.15M, pH7.2)containing 0.05% Tween 20(PBS-T) for 1.5 h at 37 ◦C. The plate was coated with recombinantly obtained extracellular matrix of antigen(0.5µg/ml of HER2 in PBS) and incubated overnight at 4⁰C. (0.5ug/ml). Blocking Buffer containing PBS with 3% non fat skimmed milk, was used to block the prepared ELISA plates, and kept in dark for 1.5hours at 37⁰C. Subsequently, Ab-CFZ-LF-NPs to the each well at different dilutions. Titrate the uncoupled antibody(1–100ng/ml) substantially to construct as standard-curve. The plate was washed repeatedly and HRP-conjugate(secondary antibody) to each well containing different concentration of serial dilutions and incubate for 1.5 h. The colorimetry reaction was revealed using enzyme substrate,ie., 3,3,5,5-tetramethyl benzidine(TMB) and the optical density was measured at 450nm using ELISA plate reader[ 38 ]. 2.7 In vitro cytotoxicity of CFZ loaded nanoparticles. The in vitro cell cytotoxicity and co localization was perfomed on SKOV 3 cell, which was obtained from the American Type Culture Collection (ATCC), USA. 2.7.1 Cell cytotoxicity studies(MTT assay): The MTT assay was performed to assess cell cytotoxic potential of CFZ-LF-NPs and FITC-Anti-Her2- CFZ-LF-NPs in SKOV-3(labelled cells for OC). Briefly, the cells at a density of 5 ×10 3 cells per ml. cells/well were seeded onto 96-well plates for overnight incubation at 37°C and 5% CO 2 . After 24 h, the medium was replaced with 100 µL of varying concentrations of Free drug(CFZ), CFZ-LF-NPs, and FITC-Anti-Her2-CFZ-LF-NPs, ranging between 10-100ng/ml, in sterile media DMEM fresh. After 8 h the growth medium was changed by fresh medium to remove the non-internalized NPs and cultured for additional 24 h. Fed the plate with complete fresh media, and was then washed with PBS(pH 7.4) at the end of growth period, and 100µl of formazan solution(MTT, 1mg/ml) followed by filtering the resulting solution with 0.2 µm membrane filter. The plates were incubated at 37°C in 5% CO 2 incubator for 4hrs. The media containing the untreated MTT was removed. Untreated cells in complete media(DMEM) were considered as negative(-)control. Triton X100 was further added in wells containing untreated cells, were considered as positive(+)control. The treatment throughout the assay was employed in triplicate Also, the cytotoxicity of NPs was evaluated in the HER2-negative ovarian cancer cell line A2780. The absorbance at 570 nm of each well was measured along-side a reference filter of 655nm using an ELISA-based microplate reader(BioRad,USA). The viable cells and %dead cell inhibition was cumulated average cell count method, wherein each absorbance of controls and samples represents the intensity of colorimetric reduction reaction of formazan take up by the treated and untreated cells, respectively. Statistical analysis of viability data was performed by 2-way ANOVA with Tukey’s multiple comparisons test. In order to determine the anti-tumor effect the IC50 of nanocarriers was also determined linear regression plot using cell Inhibition versus concentration of testing reagent[ 38 , 43 ]. 2.7.2 Cellular uptake assay: Cellular uptake of CFZ-LF-NPs, and FITC-Anti-Her2-CFZ-LF-NPs was analyzed by Confocal microscopy. Briefly, silicone supported coverslips (SecureSlip, Grace Bio-Labs) were placed in 6 well plates, then, 50,000 cells/well were cultured for 48 h with 2 mL of complete DMEM medium. The cells were then treated with free drug, as well as NPs (Free drug as control group, CFZ-LF NPs, and, FITC-AntiHer2-CFZ-LF-NPs) and Free FITC at fixed concentration determined after cytotoxicity analysis for growth period of 2 to 72 hours. Whereas, Her2 negative cell line was used as another control for the study. After this incubation period, medium was removed, and cells were washed twice with 500 µL of PBS to remove NPs that were not attached to or internalized by cells. The cells were collected for trypsinization. The trypsinized cells were neutralized by centrifuging at 1000rpm for 5 mins. The centrifuged cells were washed thrice with PBS(0.01M, pH7.4) to discard the debris or untreated dye. Cells were fixed in tissue culture dishes with 500 µL of 4% p-formaldehyde for 10 minutes, washed twice with PBS, and the coverslips were mounted on microscope slips with FluoroShield™/DAPI[ 45 ]. Slides were analyzed with an AX10 Zeiss Microscope, using DAPI and Alexa Fluor filter sets to visualize the nuclei and NPs, respectively. 2.7.3 Apoptosis and cell viability count via Flow cytometry: The seeded cells(1×10 5 )were stained with 10µl of Annexin V-FITC and PI for 10 mins at room temperature by incubating in the dark according to protocol of biosciences. The viable cell count and apoptotic cells were quantified using flow cytometer(Attune nxt, Thermofischer Scientific). The cells were quantified and observed in CellQuest solftware and the results are expressed as rate and percentage of apoptosis(late and early apoptosis)[ 24 , 46 ]. 2.8 In vivo anti-tumor efficacy studies: Ethical a pp roval and accordance All in vivo experiments on animal were approved by the Institutional Animal Care and Use Committee of IAEC Approval No : IAEC/PIPH/09/23. Moreover, these experiments were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)and ARRIVE 2.0 guidelines to ensure transparency and reproducibility (Animal Research: Reporting of In Vivo Experiments). Moreover, the experimental animals were procured from Sun Pharmaceutical Industries Ltd. , India, a licensed commercial supplier of laboratory animals(Vadodara, Gujarat) following approval of the experimental protocol from Institutional Animal Ethics Committee (IAEC) 2.8.1 Tumor distribution studies: The in vivo anti tumor effect was studies and tumor distribution studies were conducted based on the above mentioned protocol approval. Twenty healthy female athymic mice(6–8 weeks) old were commercially obtained from Sun Pharmaceutical Industries Ltd. , India, a licensed commercial supplier of laboratory animals(Vadodara, Gujarat) and housed in pathogen free environment with access to food and water. The mice were injected intraperitoneally with 1×10 7 viable SKOV-3 cells to establish tumor bearing models. After 15 days of growth, the mice were divided into three random groups of five mice: Group 1- PBS (saline control), Group 2: CFZ solution, Group 3: CFZ-LF nanocarriers. For determining tumor distribution, priorly the standard curve of CFZ in blood, and homogenized tissue samples(liver, spleen, kidney, heart, lung, uterus, small intestine, and tumor tissues) of tumor bearing mice was performed 20 µl different quantitative standard CFZ solutions (0.95, 1.9, 9.5, 19, 38, 76, and 152 µg/ml) that was added to homogenate samples (200 µl) using HPLC analysis[ 37 , 47 , 48 ]. The linear regression equation was then established based on the CFZ concentration (X) and AUC (Y) value from the HPLC test. Each mice of respective group was injected intraperitoneally with either CFZ or CFZ-LF nanocarriers with CFZ concentration of 5 mg/kg alone or in nanoparticles. Mice were anesthetized using [e.g., isoflurane / ketamine–xylazine] prior to sacrifice. At predetermined time points (1, 2, 4, 6, 12, and 24 h), 5 mice in each group were euthanized. Euthanasia was performed in accordance with CPCSEA guidelines using [method, e.g., CO₂ asphyxiation]. Further, their plasma and tissue samples were quickly collected for an HPLC test and then calculated based on the linear regression equation obtained as mentioned above to determine the CFZ concentration in the samples[ 24 , 49 ]. Based on concentration of drug reached varied organs was quantified using HPLC based standard curve, the tumour distribution in separate homogenized organs was determined. 2.8.2 Tumor Growth inhibition study(anti-tumor effect): The tumor bearing mice were randomly divided in four groups(6/group): Group 1: PBS(control), Group 2: free CFZ, Group 3: FITC-CFZ-LF-NPs, and Group 4: FITC-Anti-Her2-CFZ-LF-NPs. A single dose of CFZ alone and in nanocarriers was intravenously administered to each mouse and saline solution was used as a control. Each mouse received a treatment of 120 µL via intraperitoneal injection once a week for two weeks. All samples were freshly prepared and filtered prior to injection[ 37 , 50 ]. The mice were inspected daily and the overall body condition and body weight were measured. The tumor sizes and animal body weights were then measured every 3 days. After thorough observation, at the endpoint of treatment, the mice were euthanized and organs(liver, spleen, kidney, heart, lung, uterus, small intestine, and tumor tissues) were collected and placed in 10% formalin for Hemotaxylin & Eosin (H&E) followed by immunohistochemistry was performed. Moreover, the average tumor volume was calculated that was used to determine the effectiveness of treatment. Statistical analysis : The results were reported as mean ± standard deviation. Student’s t-test in Excel was used to analyse the statistical significance of the tumour growth inhibition. A value of p < 0.05 was considered statistically significant. 3. Result and discussion 3.1 Nanoparticles characterization and evaluation: ‘Carfilzomib loaded lactoferrin nanoparticles’ (CFZ-LF-NPs), and anti-Her2 nanoconjugates(Anti-Her2-CFZ-LF-NPs) were ascertained for their particle size and zeta potential as shown in Fig. 1 a and b , by using dynamic light scattering (DLS) in Malvern Z90 zetasizer equipped with internal laser. The particle size distribution, PDI, and zeta potential was found to be in the range of 80 to 250 nm, 0.2 to 0.7 and + 2.01 to + 10.45 for CFZ-LF-NPs that indicates the heterogeneously distributed polydisperse nanoparticulate system. Figure () delineating the particle size pattern of distribution and zeta potential of optimal composition of CFZ-LF-NPs, which was found to be 80.39 ± 0.014 nm and + 14.58 zeta potential(Fig. 1 a). This indicates the narrow uniform size distribution with 0.227 ± 0.032 PDI as well as net positive surface charge indicates that lactoferrin is positively charges below isoelectric point, delineating suitable stability that was anticipated for lactoferrin (with pI 8 )[ 43 , 51 ]. However, after conjugating with Anti-Her, the particle size of Anti-Her2- CFZ-LF-NPs was found to be 240 ± 0.224nm and 0.357 ± 0.412PDI as well as zeta potential was observed to be 26.85 ± 0.147(Fig. 1 b ) , indicating the potential stability of formulation. ‘Carfilzomib loaded lactoferrin nanoparticles’ (CFZ-LF-NPs) synthesize by Desolvation, subjected 1 H NMR spectroscopy The Fig. 2 a, b, c, d, showing the NMR spectra of, CFZ- LF-NPs, free EDC NHS, AntiHer2-CFZ-LF-NPs before and after dialysis to confirm the removal of unreacted residues. The multiple signals observed between 3.2 to 4.8 ppm were assigned to the resonance of methylene protons -Ch3-, depicting the evident spectrum peaks corresponding to LF. In addition. signals at 2.78 ppm assigned to aliphatic protons -CH2-NH-C = O linked secondary amine corresponding to peptide chain of LF. Aromatic amine, and peaks of deshielded aromatic proton at downfield between, as observed in free LF, which can be also seen in NMR spectra of CFZ-LF-NPs. The NMR spectrum were acquired as shown in Fig. 2 , showing the conjugation of AntiHer2 and CFZ-LF-NPs. The NMR signals in CFZ-LF-NPs, showing the chemical shift of methylene protons at up-field can be evidently observed in Ab-CFZ-LF-NPs at 4.96 ppm(Fig. 2 a). The shift may be due to electronegativity of neighbouring groups belonging to Anti-Her2. Moreover, antibodies are much high in molecular weight, so clusters of signals may be observed that may results in noise interpretation. However, the small signals at 5.2 ppm, attributes to CONH formation, confirming the conjugation(Fig. 2 c and d ). Also, compared with free EDC- NHS(Fig. 2 b) showing signals in methylene regions, while no signals were acquired in AntiHer2-CFZ-LF-NPs(Fig. 2 d). The removal of isourea(byproduct of EDC-NHS reaction) was confirmed by NMR of Ab conjugates before and after dialysis(Fig. 2 c and d ). The signal observed between 2.5 to 3 attributes to methyl group of isourea byproduct ( Fig. 2 b and c ), as shown in NMR of AntiHer2-CFZ-LF-NPs(before dialysis), and the intensity of which is completely reduced after dialysis as shown in Fig. 2 d). Moreover, the intensity of signals of succinimide ring attributed at 2.78 ppm were completely reduced after dialysis in AntiHer2-CFZ-LF NPs, as compared to evident peaks in NHS(Fig. 2 b, c and d ) with high intensity. This confirmed the removal of byproducts and unreacted EDC-NHS and its intermediate product[ 52 ]. The antibody conjugation labelled with FITC dye can be emerged as cancer theragnostic tool for biological imaging. Moreover, FITC have the capability to react with amine reactive groups on proteins and antibody inside and outside the body, hence FITC(0.1mg/ml) was used for conjugated with Anti-Her2-CFZ-LF-NPs. The fluorescence spectrophotometer(Jasco) was used for determining the FITC content in nanoconjugates to demonstrate a fluorophore intensity for biological imaging purpose[ 52 , 53 ]. The substantial rise in fluorescence intensity(FI) with concurrently augmenting concentration of nanoconjugates, encompasses the FITC conjugation of nanoconjugates was compared with Free FITC as shown in Fig. 3 ). The experiments were conducted in triplicate and the concentration of FITC in nanoconjugates was calculated using the average absorbance, which resulted in 18.14 µg/ml and 19.04 µg/ml concentration in FITC-AntiHer2-CFZ-LF-NPs, thereby confirming more than 90% conjugation with R-square values,i.e., r2 = 0.9901(Fig. 3 ). Therefore, high linearity near to 1 entail less than 2%RSD with high FITC conjugation. Surface morphological examination of protein nanoconjugates(CFZ-LF-NPs and FITC-Anti-Her2-CFZ-LF NPs) was assessed by Transmission emission scanning microscopy as shown in Fig. 4 a and b , which demonstrate the spherical shape of lactoferrin nanoparticulate system uniformly distributed within size range of 80 to 200 nm. The entrapment efficiency of CFZ was calculated by reconstituting the freeze-dried product as per the above-mentioned procedure and centrifuged at 10,000 rpm, where methanol or acetonitrile was used as a diluent to separate the un-entrapped drug in the supernatant, which was further analysed for drug entrapment by HPLC[ 44 , 54 ]. The entrapment efficiency of drug in CFZ-LF-NPs was found to be 86% ± 0.289 with %DL of 14.14 ± 0.521%. This was determined by using a validated HPLC method for carfilzomib estimation at 210 nm wavelength, as shown in supplementary Figure S1 . Sharp chromatogram of isolated supernatant samples containing free drug separated at 7.45 RT in HPLC( supplementary Figure S1 ). However, disappearance of CFZ peak with HPLC chromatogram of LF (11.2 min RT) having sharp resolution shown in supplementary Figure S1 confirmed the drug entrapment. The CFZ-LF-NPs were subjected to in vitro drug release using Franz diffusion cell, at physiological condition (pH 7.4) and the conditions prevalent in tumor (pH 5.5). The in vitro drug release curve is shown in Fig. 5 . In vitro drug release was investigated by developed and validated HPLC method. The results obtained from drug diffusion study of CFZ-LF-NPs at pH 7.4 was found to be 65.258 ± 0.382% (up to 8 days) as shown in Fig. 5 . It could be concluded that the invented formulation was able to prolong the drug release continuously up to a week avoiding frequent application of the formulation. The final formulation of CFZ-LF-NPs had shown fast and highest drug release of 91.585 ± 0.017% (Fig. 5 ) at tumor pH of 5.5 up to 96 hours, and sustained the drug diffusion at physiological condition till 8 days. This observation delineates pH sensitive drug release by the formulation (CFZ-LF-NPs) that was one of the objectives to prolong the retention of the active drug at physiological pH and enhance the diffusion at tumor microenvironment (pH 5.5). Whereas, untreated drug was released shortly within 72 hrs. This concluded that the drug conjugate of protein nanoparticles is capable of retaining the drug physiologically and sustained the diffusion with pH sensitive response at acidic pH (5.5). The in vitro drug release was quantified by developed and validated HPLC method for estimation of carfilzomib at 210 nm as shown in supplementary Figure S2 , with chromatogram taken at variable time in hours. The long term stability study was employed to evaluate and observe the changes in the quality, safety, appearance, compatibility of the formulation when exposed to various temperature conditions and to observe how the parameters keep on changing over the time of storage. Stability studies revealed that the nanoparticles stored over a storage of 6 months when kept at both refrigerated temperature as well as room temperature, whereas storage at 4–8°C ± 2°/45%±5% RH resulted with minimal changes in size and %EE as compared to 25°C ± 2°C/65 ± 5% RH, hence this delineates the stability of protein nanoparticles more under refrigerated conditions ( supplementary Table S1 ). Thus, purpose is to obtain a long-term storage of the formulation to maintain the shelf life the formulation was successfully employed. 3.2 ELISA A test for examining anti-Her2 concentration in nanoconjugates (FITC-Anti-Her2-CFZ-LF-NPs): ELISA results( supplementary Figure S3 ) revealed that successful conjugation of Anti-Her2 with rise in optical density at higher concentration of FITC-Anti-Her2 conjugated Lactoferrin. Nanoparticles. Highest optical density achieved 600 ng/ml of FITC-Anti-Her2 conjugated Lactoferrin Nanoparticles indicates irrefutable in its targeting affinity but also confirms successfully employed conjugation. Substrate TMB oxidizes to TMB + 2 when it reacts with HRP-conjugate and after adding the stop solution (acid) the colour changes from blue to yellow due to HRP degradation and acidification of TMB. The entire enzymatic reaction reveals estimation of target protein. The intensity of colour indicates the amount of antibody conjugated with target antigen via enzyme substrate affinity towards the primary and secondary antibody. The optical density inclined with increasing concentration that can be inferred from supplementary Figure S3 . The optical density was found to be within limits when compared with standard. However more than 60 ± 0.225% of AB conjugation was recovered, depicting the coupling of anti-Her(1 mg/ml) with CFZ-LF-NPs [52,55,56]. 3.3 In vitro cytotoxicity of CFZ loaded nanoparticles. 3.3.1 Cell cytotoxicity studies(MTT assay): The cell culture examination was erformed for determination the percentage cell inhibition(MTT assay) of nanoformulation in labelled SKOV-3 cells, including CFZ-LF-NPs, and FITC-AntiHer2-CFZ-LF-NPs and Carfilzomib alone(control)[ 45 , 52 ]. The MTT assay for determining the cell cytotoxicity was assessed till 72 hours of treatment. It was observed that Anti-Her2 nanoconjugates(FITC-AntiHer2-CFZ-LF-NPs) showed more than 90% of cell inhibition,i.e., 95.72 ± 1.44% at 5nM concentration which is 2-fold enhanced inhibition as compared to free drug. Whereas, CFZ-LF-NPs shown 66.83 ± 1.4 % cytooxicity that is less than FITC-AntiHer2-CFZ-LF- NPs. Unlikely, plain drug resulted in least cell cytotoxicity with 45.60 ± 2.15% even at the highest concentration of 25nM as shown in Fig. 6 . Therefore, the results clearly unravels high efficacy of Anti-Her2 nanoconjugates(FITC-AntiHer2-CFZ-LF-NPs), as antibody conjugation serves as dual therapeutics against cancer with target specific uptake, as compared to CFZ-LF-NPs (non-conjugated) 60% cell inhibition thereby depicting target specificity for inducing cell death. Hence, the nanoconjugates can be emerged as a futuristic nano-cargo for cancer theragnostic in molecular imaging. Additionally, anti-tumor effect was evaluated in vitro by determining IC 50 values. The nanoparticles and nanoconjugates resulted with 11.167nM(AntiHer2-CFZ-LF-NPs), and 13.447nM(CFZ-LF-NPs) of IC50, thereby exhibiting a 2-fold reduction in inhibitory concentration of drug in nanoconjugates as compared to Carfilzomib alone as single drug therapeutics with 25.231nM IC50. Thereby, indicating nanoformulation enhanced therapeutics of drug with minimum bare concentration for cell inhibition. 3.3.2 Cellular uptake assay: The cellular uptake was ascertained using dye staining under confocal microscopy to determine the cell internalization and apoptosis of CFZ-LF-NPs and FITC-Anti-Her2-CFZ-LF-NPs that was compared with non treated cells(PBS) as negative control and free CFZ as positive control. The live cells were stained using DAPI and dead cells were stained with propidium iodide using Annexin-V kit[ 52 ]. The results revealed highest cellular uptake of FITC-Anti-Her2-CFZ-LF-NPs as compared to CFZ-LF-NPs in Her2 labelled SKOV-3 cells due to enhanced target specificity of Anti-Her2 conjugated lactoferrin nanoparticles as observed in Fig. 7 a, b, c, d. Moreover, iron de-efficiency is highly overexpressed in proliferating cells, thereby iron containing lactoferrin synergistically contributed in cell internalization. Figure 7 d unravels tumor necrosis was observed in Anti-Her2 treated cells after 72 hours, with red chromatin nuclei (PI stained cell death). The red stained chromatin fibrosis in cells is high observed in FITC-AntiHer2 -CFZ-LF-NPs as compared to CFZ LF-NPs. Hence, LF NPs highly taken up via iron de-efficient cancer cells, whereas Anti-Her2 conjugation prominently shown high stained dead cells as shown in Fig. 7 c and d . Therefore, the study encompasses the dual cancer therapeutics of using Anti-Her2 with carfilzomib loaded LF NPs with target specificity against OC cancer phenotypes. 3.3.3 Apoptosis and cell viability count via Flow cytometry: The SKOV-3 cells were treated with CFZ(5nM), CFZ-LF-NPs(5nM), and FITC-AntiHer2-CFZ-LF-NPs(nM) (Fig. 8 a, b, c) followed by 24 cell culturing and staining with Annexin-V and PI(propidium iodide) kit for determining the apoptosis in cell population in different time intervals. The Annexin-V having the ability to interact with phosphatidylserine of a live cell with initial stage of apoptosis and PI binds penetrates in dead nuclei of cells for labelling dead cells. However, the apoptotic cell population can be identified by separate particles in black dot diagram as shown in Fig. 8 with flow cytometry results at different time intervals(48 and 72 hours), in which there are 4 quadrants. Amongst the four quadrants, the lower left quadrant(Q3) depicts viable cells,i.e., the cells which are negative for PI and Annexin uptake, while the cells which are positive for Annexin-V and negative for PI are demonstrated as early apoptotic cells as distributed in lower right quadrant(Q4). In this early apoptosis, the cell membrane begin to shrink resulting in increasing intensity of side scatter in flow cytometer. The cells having positive staining of both PI and Annexin-V are late apoptosis/necrotic cells shown in upper right quadrant(Q2), while the cells which are PI positive and AnnexinV-negative are necrotic cells shown in upper left quadrant(Q1). This can be inferred from figure, depicting the clustered of cell population in control, that reduced and the separate cell entity increased in CFZ-LF-NPs, FITC-AntiHer2-CFZ-LF-NPs, thereby showed increase in apoptosis percentage with cell shrinkage. The apoptosis percentage of CFZ, CFZ-LF-NPs, and FITC-AntiHer2-CFZ-LF-NPs was observed to be increased from 9%, 27%, and 39% to 23%, 64.5%, and 70.8% in 72 hours of treatment. This increment from 24 to 72 hours treatment, demonstrate a timely-dependent apoptosis. Noticeably, CFZ alone shown incremental rise in apoptosis in initial 12 hours after which a steep decline in rise was observed Fig. 8 a. As anticipated, FITC-AntiHer2- CFZ-LF-NPs showed the highest late apoptosis compared to CFZ-LF-NPs as observed in Q2(Fig. 8 b and c ), and cell populations in Q1, thereby depicting a necrosis followed by late apoptosis indicating high cell death. However, a shift from high intensity of side scatter to front scatter was observed in FITC-AntiHer2-CFZ-LF-NPs(Fig. 8 ). As reduced side scatter(SSC) and increased(FSC) determines a complete cell shrinkage resulting late apoptosis/necrosis. Moreover, FITC-AntiHer2-CFZ-LF-NPs showing completely inclined FSC, thereby demonstrating high apoptosis due to dual synergistic effect of anti-Her2 and CFZ in SKOV-3 cell population. Moreover, the increase in apoptosis percentage induced in cells treated with CFZ-LF-NPs, FITC-AntiHer2-CFZ-LF-NPs, encompasses an enhanced drug efficacy through aid of protein nanoconjugates[ 45 , 52 ]. 3.4 In vivo anti-tumor efficacy studies: 3.4.1 Tumor distribution studies: The CFZ levels of plasma and other tissues were studied at different points delivered from conjugated(CFZ-LF-NPs and non-conjugated(FITC-AntiHer2-CFZ-LF-NPs nanoparticles as shown in Fig. 9 . Firstly, peak extended time delineating absorbed concentration in all nano carrier groups were compared with the free drug(CFZ) in different areas of tissues including tumor tissues as shown in Fig. 9 . These results again showed slow drug release in nanocarrier groups with gradual rise in concentration in tissues, whereas initial controlled release effect. While, free drug concentration was high initially, that showed steep decline with less penetration to deep tissues(Fig. 9 ). Most importantly in tumor tissues, the CFZ concentration in FITC-AntiHer2-CFZ-LF-NPs(Fig. 9 ) at 12 hours were much higher than nonconjugated CFZ-LF-NPs and free CFZ as shown in Fig. 9 , while there was a negligible statistical difference between the latter two groups in other tissues. These results in Fig. 9 clearly delineate the AntiHer2 and LF mediated active targeting efficacy of FITC-AntiHer2-CFZ-LF-Nps at target tumor site with high penetration and uptake as compared to normal tissues[57]. This indicates less plausible dose dependent toxicity in normal tissues with nanoconjugates. However, except for liver, uterus, and spleen CFZ-LF-NPs has greater uptake in other tissues, that delineated not target specificity. Moreover, in Heart, Kidney, and lung significantly low levels of CFZ in FITC-AntiHer2-CFZ-LF-NPs was observed. This indicates improved pharmacological effect of lactoferrin nanoconjugates of carfilzomib by preventing drug accumulation in non-target tissues, thereby reducing the toxicity. 3.4.2 Tumor Growth inhibition study: The final tumor size in treated mice were notably reduced in FITC-AntiHer2-CFZ-LF-NPs as observed in Fig. 10 , while there was less statistical difference between free CFZ and CFZ-LF-NPs groups(p < 0.005). The final tumor size in FITC-AntiHer2-CFZ-LF-NPs treated mice was found to be 312.67 ± 41.45 mm 3 ), much smaller than those treated with CFZ-LF-NPs(487.13 ± 32.12 mm 3 ) and free CFZ(508.56 ± 39.33 mm 3 ) groups as well as controls (805.49 ± 55.23 mm 3 ) as shown in Fig. 10 . All the treatment groups exhibited significant antitumor effect as compared to the control groups(p < 0.05)[57]. The tumor growth curve showed that FITC-AntiHer2-CFZ-LF-NPs has much stronger antitumor effect as compared to other groups of CFZ-LF-NPs and free CFZ (Fig ). The inhibitory rate based on tumor volume were 61.88% in FITC-AntiHer2-CFZ-LF-NPs, that is 2-fold times higher than in CFZ-LF-NPs(27.23%) and free CFZ(23.12%). Negligible changes in body weight in all the four groups(p < 0.05) indicated no obvious side effects in the nano-carriers groups. Conclusion The research work emphasizes surface functionalized carfilzomib loaded lactoferrin nanoparticles using different biomarkers for molecular recognition such as anti-Her2(Herceptin). CFZ was chosen for several reason concerned with its potential clinical outcomes, irreversible action, and motif to prevent its peptide mediated clearance and enhance its prolongation. However, protein are more susceptible of protein corona formation with serum proteins body. Therefore, to prevent this rigidizing its surface with molecular tag can be potential approach. The lactoferrin nanoparticles were functionalized with anti-Her2 using EDC and NHS based carbodiimide chemistry. The above discussed formulations synthesized and were evaluated for anti-tumor activity in SKOV-3 cells for determining the cell cytotoxicity, cellular uptake, apoptosis mechanism, and viable cell count using plate reader, confocal imaging, and flow cytometry. The results of these studies revealed 2 fold times tumor inhibition in mice treated with nanoconjugates(FITC-AntiHer2-CFZ-LF-NPs) with same dose that of free CFZ and CFZ-LF-NPs(5 mg/kg), underscoring potential target specific treatment. This antitumor effect in such metronomic dose can be a promising approach in cancer treatment. There was no reccurence was observed in treated mice indicating improved survival in vivo . The prominent results and observation indicates beyond the scope, that the developed biomarkers tagged carfilzomib loaded lactoferrin nanoparticles may emerge as spy-catching tool in combination with therapeutics for dual targeting with enhanced stability for intravenous administration in cancer theranostics. Declarations Ethics Approval and Consent to Participate Not applicable. Ethical approval and accordance Human and Animal Rights No human participants were involved in this study. All in vivo experiments on animal were approved by the Institutional Animal Care and Use Committee of IAEC Approval No: IAEC/PIPH/09/23. Moreover, these experiments were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)and ARRIVE 2.0 guidelines to ensure transparency and reproducibility (Animal Research: Reporting of In Vivo Experiments). Data availability statement The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethics and consent to participate The experimental animals(athymic mice) were procured from Sun Pharmaceutical Industries Ltd. , India, a licensed commercial supplier of laboratory animals(Vadodara, Gujarat) following approval of the experimental protocol from Institutional Animal Ethics Committee (IAEC). As the animals were obtained commercially from an authorized supplier, no additional institutional permission or owner consent was required beyond approval from the Institutional Animal Ethics Committee (IAEC). The relevant details have been updated in the manuscript. Consent for Publication Not applicable. Availability of data and materials: This contribution is not included with any extra files. Conflict of Interest The authors declare no conflict of interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8494829","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":587326166,"identity":"a8fa9137-4a84-4e09-a566-49075f527900","order_by":0,"name":"Drishti Panjwani","email":"","orcid":"","institution":"Parul University","correspondingAuthor":false,"prefix":"","firstName":"Drishti","middleName":"","lastName":"Panjwani","suffix":""},{"id":587326185,"identity":"a55baac6-2459-4dea-b42f-6d6afcfaa72e","order_by":1,"name":"Asha Patel","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIie2Pv2qDUBSHj1zQRXG1hOYNChYhIRDyLPcgOBZKlwxBhECySGcLJX0Fu7j2yoFMBdeOXnwBp5Cp9AYy5opjh/txlvPng/MDMBj+Ky0ABwaczmvVOQBWNnjPLveqHJAdfAO4bLTiW11k7UYoc6epW75Jn/wtSybPhyV+MVbLAlbTB423yGMW8iO9BGQnk7cqwZzZ8WMJcTQTt5VQxHbAbYEZuUngVaQUd3bXgsBKpzSdc+a/KX6Qfwq89zHKT2wD7hiWpH7xsqtSDiiLoosCfCX8JEBZHJMoJ5WlCPVZ5j7Kvj+leGgEF/1meb/fb2uZr1dTbfzbY+18eGUwGAyGK3/LsWIAdBluXgAAAABJRU5ErkJggg==","orcid":"","institution":"Parul University","correspondingAuthor":true,"prefix":"","firstName":"Asha","middleName":"","lastName":"Patel","suffix":""},{"id":587326198,"identity":"93bed60c-0638-4682-86db-be1d03af0f36","order_by":2,"name":"Shruti Patel","email":"","orcid":"","institution":"Parul University","correspondingAuthor":false,"prefix":"","firstName":"Shruti","middleName":"","lastName":"Patel","suffix":""},{"id":587326200,"identity":"1b22e37f-3a72-4c65-b205-5da328ec14b5","order_by":3,"name":"Nishabh Kushwaha","email":"","orcid":"","institution":"Parul University","correspondingAuthor":false,"prefix":"","firstName":"Nishabh","middleName":"","lastName":"Kushwaha","suffix":""},{"id":587326201,"identity":"069b9f04-12dd-49ae-9f9e-33d2aa461088","order_by":4,"name":"Viral Patel","email":"","orcid":"","institution":"University of Alberta","correspondingAuthor":false,"prefix":"","firstName":"Viral","middleName":"","lastName":"Patel","suffix":""},{"id":587326207,"identity":"171e559b-0355-41a6-a1fd-72044d93044e","order_by":5,"name":"Priyanka Ahlawat","email":"","orcid":"","institution":"Parul University","correspondingAuthor":false,"prefix":"","firstName":"Priyanka","middleName":"","lastName":"Ahlawat","suffix":""},{"id":587326211,"identity":"44d58242-ea0b-48e2-8f60-29cb0b1e4222","order_by":6,"name":"Vibhuti Agrahari","email":"","orcid":"","institution":"Oklahoma State University","correspondingAuthor":false,"prefix":"","firstName":"Vibhuti","middleName":"","lastName":"Agrahari","suffix":""},{"id":587326214,"identity":"314ff63c-4881-47b0-b9aa-61e394ded4fc","order_by":7,"name":"Mange ram yadav","email":"","orcid":"","institution":"Parul University","correspondingAuthor":false,"prefix":"","firstName":"Mange","middleName":"ram","lastName":"yadav","suffix":""}],"badges":[],"createdAt":"2026-01-01 11:23:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8494829/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8494829/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102239185,"identity":"6cdae409-f858-42db-849c-237bcb0d1122","added_by":"auto","created_at":"2026-02-09 16:43:24","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":147040,"visible":true,"origin":"","legend":"\u003cp\u003eParticle size and zeta potential determination; a) CFZ-LF-NPs and b) FITC-AntiHer2-CFZ-LF-NPs.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/b523972b86c5dff0813b309f.jpg"},{"id":102239188,"identity":"4e327832-2ad4-4cbe-80db-a67b89e6e7b7","added_by":"auto","created_at":"2026-02-09 16:43:24","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":79680,"visible":true,"origin":"","legend":"\u003cp\u003eNMR spectroscopy for determining the structural integrity CFZ-LF-NPs, AntiHer2-CFZ-LF- NPs; a) CFZ-LF-NPs, b) EDC, c) AntiHer2-CFZ-LF- NPs before dialysis, and d) AntiHer2-CFZ-LF- NPs after dialysis for removal of unreacted residues.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/0cafd1ddfff811e5f0107dc7.jpg"},{"id":102239193,"identity":"f8a38077-0edb-4fd8-8651-8da7b45a8041","added_by":"auto","created_at":"2026-02-09 16:43:24","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":58987,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence spectroscopy of FITC-AntiHer2-CFZ-LF-NPs\u003c/p\u003e","description":"","filename":"Figure3..jpg","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/c420c74dde10e257ade356d5.jpg"},{"id":102239184,"identity":"a57584ad-4d0a-47ed-8cb6-50054ba88b69","added_by":"auto","created_at":"2026-02-09 16:43:24","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":336794,"visible":true,"origin":"","legend":"\u003cp\u003eTEM morphological image of a)CFZ-LF-NPs and b) \u0026nbsp;AntiHer2-CFZ-LF- NPs\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/aa1af9450f8bfb9d9c291baa.png"},{"id":102297446,"identity":"96984131-dc9c-42fc-87b5-5df0185943c1","added_by":"auto","created_at":"2026-02-10 10:27:27","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59014,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e drug release plot of %CDR vs Time\u003c/p\u003e","description":"","filename":"Figure5..jpg","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/dcfabda0601ad5acbbbe3973.jpg"},{"id":102297174,"identity":"4a504f1a-9485-4775-baf0-456f22d4f81a","added_by":"auto","created_at":"2026-02-10 10:26:16","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":59847,"visible":true,"origin":"","legend":"\u003cp\u003e72 hours %cell inhibition(MTT assay) of SKOV-3 cells after treatment with free CFZ, CFZ-LF-NPs, and, FITC-CFZ-AntiHer2-LF-NPs\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/458240f5d31b498a30c5a728.jpg"},{"id":102298672,"identity":"c99336ef-2eb9-48d7-ac81-bf6b6e927697","added_by":"auto","created_at":"2026-02-10 10:57:56","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":103700,"visible":true,"origin":"","legend":"\u003cp\u003eCellular internalization for determining live and dead cells via staining using Annexin-V Kit: A PBS, B Free CFZ, C CFZ-LF-NPs, and D FITC- AntiHer2-CFZ-LF-NPs.\u003c/p\u003e","description":"","filename":"Figure7..jpg","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/a5e6c1bb8d9efed78b694c03.jpg"},{"id":102297055,"identity":"e8655309-703a-4d81-8092-e0b3b1dff01c","added_by":"auto","created_at":"2026-02-10 10:25:24","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":130744,"visible":true,"origin":"","legend":"\u003cp\u003eApoptosis and viable cell count: \u0026nbsp;a) PBS , b) CFZ-LF-NPs, and c) FITC-AntiHer2-CFZ-LF-NPs by flow cytometry.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/23f9f917cc0a5c2986f3bbee.jpg"},{"id":102239194,"identity":"4450bed6-ee4c-475f-89fa-3d1f9c075071","added_by":"auto","created_at":"2026-02-09 16:43:24","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":198023,"visible":true,"origin":"","legend":"\u003cp\u003eTumor distribution of CFZ in different tissues including tumor tissues at various time points post treatment with free CFZ, CFZ-LF-NPs and AntiHer2-CFZ-LF-NPs\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/7e7b5f9b73132b822d175a35.jpg"},{"id":102297294,"identity":"2d4dac02-4241-44ed-a2d4-79edbe096099","added_by":"auto","created_at":"2026-02-10 10:26:51","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":58802,"visible":true,"origin":"","legend":"\u003cp\u003eAnti tumor effect observed in nude mice post treatment with CFZ-LF-NPs and AntiHer2-CFZ-LF-NPs\u003c/p\u003e","description":"","filename":"Figure102.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/5c9f480cdc84078599a0c1c9.jpg"},{"id":102301237,"identity":"28b7b193-9b19-429b-81e9-9944152f85db","added_by":"auto","created_at":"2026-02-10 11:20:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2415020,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/f321401e-8ace-4794-80b1-e7a592ac5a00.pdf"},{"id":102239191,"identity":"e095e4c3-67c7-484c-b692-43b2fdd41ba1","added_by":"auto","created_at":"2026-02-09 16:43:24","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2291572,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-8494829/v1/54fd7780c6719cd5808ae65a.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immunomodulatory and Antitumor Activity of Surface-Functionalized Lactoferrin Nanoparticles in Ovarian Carcinoma","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eOvarian cancer(OC) is one of the most common gynaecological cancer with high morbidity, and prevailing cause of female community leading cause of death worldwide[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. This is majorly because of its asymptomatic nature that leads to advanced stage diagnosis and thereby resulting in malignant ascites with poor prognosis[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. According to GLOBOCAN, the incidence rates for OC increased to 315,949 with 300000 deaths worldwide[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Despite of numerous efforts that have been made using traditional treatments including chemotherapy, in which surmounting undesired off-target effects, recurrence, immune-suppression and emergence of multi-drug resistance remains a challenge[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Therefore, this have created an urge for employing precise and selective targeting of heterogenous tumor markers, immune modulation, and targeting proteins that are deranged in ovarian cancer.\u003c/p\u003e \u003cp\u003eProteomics is a promising approach in targeting the proteins that are deranged in cancer[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Hence, precise dysregulation of Ubiquitin proteosome pathway can be a potential target in cancer, wherein terminating the protein degradation leading to ER stress followed by cyclin pathway mediated cell death[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Carfilzomib is a front line proteosome inhibitor, with high potency to bind irreversibly at active site of immune proteosome and inhibit immune checkpoints. Despite of potential clinical outcomes, it is associated [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e] with some limitations including rapid clearance(\u0026lt;\u0026thinsp;30mins), poor biostability results in incomplete dose maintenance at tumor site, and low solubility[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To mitigate these shortcomings, combining therapeutics of such immune checkpoint inhibitors like carfilzomib with functional nanomaterial that can enhance the targeting affinity, and drug retention at target site[\u003cspan additionalcitationids=\"CR17 CR18 CR19 CR20 CR21\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eProteins like lactoferrin have garnered superior attention as nanocarriers due to its multi-functional role as anti-inflammatory, anti-microbial, immune modulatory, and ability to employ iron homeostasis[\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Lactoferrin is iron containing glycoprotein of transferrin family with potential ability to scavenge free iron at inflamed sites, that enables iron deficiency in cancer cells, thereby contributing to cell death[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In contrast to synthetically derived polymers releasing toxic metabolites upon degradation, lactoferrin based proteins as natural building blocks get metabolized into amino acids which can be used by the body tissues. Additionally, proteins and iron remain in high demand by proliferating cancer cells, thereby overexpresses transferrin receptor that favor receptor mediated uptake of cargoes containing lactoferrin nanocarriers[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. These multifunctional role makes lactoferrin an ideal drug carrier that not only carries the drug to target site but also synergistically contributes with drug in therapeutically deceiving the cancer cells. However, proteomics delivery endures interaction with serum proteins, thereby increasing a surge for progressive advancement in genetic and molecular modalities in development of target specific biomarker-driven therapy[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR29 CR30\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this context, we developed Anti-Her2 tagged carfilzomib loaded lactoferrin nanoparticles as Her2 specific functional nanomaterials for immune-biotherapeutics, designed to integrate immune-checkpoint inhibition with antibody-mediated tumor targeting in ovarian cancer. Moreover, lactoferrin is capable of targeting transferrin receptor overexpressed on cancer cells requiring iron for proliferation. However, concerning with tumor heterogenicity in OC phenotypes, modifying the programmable assembly of lactoferrin by altering the surface with active ligand molecules for active targeting facilitates dual targeting and combined therapeutics. Based on reports, ameliorating multifunctional targeting by employing molecular tagging on proteins like lactoferrin that can not only carry the drug to target site but also synergistically contribute in enhancing apoptosis in cancer cells[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this work, AntiHer2 was conjugated with nanoparticles using carbodiimide chemistry[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], that was confirmed using NMR and ELISA followed by detailed evaluation of anti-tumor effect in SKOV-3 cells. The findings of our research suggests that our developed formulation may emerge as a molecular docks comprising nanomedicine with significant application in biosensing,bio imaging as well as immunotherapy-based theragnostic.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemicals:\u003c/h2\u003e \u003cp\u003eCarfilzomib (CFZ) (99.8% pure) was obtained as a gift sample from BioconLtd. Lactoferrin(pharmaceutical grade) and Anti-Her2 was procured from sigma Aldrich, Mumbai. Desolvating solvents like methanol, doubled distilled water, and acetone were procured from chem dyes, India. Other chemicals utilized from chemical synthesis of nanoconjugates of carfilzomib like 1-Ethyl-3-(3 dimethylaminopropyl)carbodiimide(EDC), N-hydroxysuccinimide(NHS) N,N-Dicyclohexylcarbodiimide Glutaraldehyde, were supplied by Sigma Aldrich, Mumbai. HPLC grade solvents were procured from Qualikems, India. SKOV-3 cells were procured from ATCC, USA. Other materials used for cancer cell culture studies such as bioimaging molecular tag(FITC), 4',6-diamidino-2-phenylindole (DAPI), Annexin-V kit, Propidium iodide, Fetal bovine serum(FBS), and Dulbecco\u0026rsquo;s Modified Eagle Medium(DMEM) were procured from Sigma Aldrich Mumbai. ELISA kit, Cell counting Kit(CCK), cell culture well plates, and trypsin were obtained from Thermofischer Scientific(Shanghai China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Synthesis of lactoferrin nanoparticles.\u003c/h2\u003e \u003cp\u003eCarfilzomib loaded lactoferrin nanoparticles(CFZ-LF-NPs) were fabricated using desolvation technique, which is based on the principle of self-assembly. Firstly, the LF was dissolved in double distilled water to prepare the aqueous solution, and then the prepared solution was filtered through the 0.45\u0026micro;m pore size of Millipore membrane. Further, the pH of aq solution was adjusted to 5 using 0.1N HCl. Further, the drug carfilzomib was dissolved in binary mixture of desolvating agent solution(ethanol:water) in a ratio of 1:1 to 3:1%w/v. The desolvating solution is then added gradually dropwise in aq solution of lactoferrin. The principle of self-assembly is based on protein folding/unfolding transition via disturbing the native environment(pH, temperature, and charge), that enables the exposure of hydrophobic domains for ameliorating [\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] physical encapsulation of drug. The resultant nanoparticles were further chemically crosslinked by adding varied concentration of glutaraldehyde (0.05\u0026ndash;0.1%) into the turbid suspension to rigidize lactoferrin nanoparticles by stirring the nanoparticles to prepare crosslinked carfilzomib loaded lactoferrin nanoparticles. The resulting mixture was subjected for centrifugation ate 5000rpm to eliminate the un-entrapped drug. The resulting supernatant was extracted and the pellets were repeatedly washed with deionized water in order to obtain \u0026lsquo;Carfilzomib loaded lactoferrin nanoparticles\u0026rsquo;(CFZ-LF-NPs). The nanoparticles were further freeze-dried using Epsilon alpha 1\u0026ndash;4 LSC(Martin Christ, Germany) according to the developed and optimized recipe for lyophilization\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Fabrication of Anti-Her2 conjugated lactoferrin nanoparticles.\u003c/h2\u003e \u003cp\u003eThe surface modification of CFZ-LF-NPs was carried out using EDCNHS carbodiimide chemistry. Briefly, 4 mg EDC(0.4%w/v) was dissolved in 1 ml of PBS(0.1M, pH 5.8) containing 1 mg NHS, followed addition of 10\u0026micro;l of anti-Her2(1mg/ml in 0.1M PBS, pH 7.4) forming a suspension under continuous stirring till 30 mins. Further, CFZ-LF-NPs(10mg) were reconstituted in 1 ml of PBS (0.1M pH5.8) and were added in above activated anti-Her2 solution. The resulting solution was allowed to stir continuously for 4 hours followed by incubation at freezing temperature(4⁰C) for 1 hour. Then, resulting was further centrifuged at 10,000 rpm, 4⁰C in a triplicate manner to remove excess EDC and unconjugated residues [\u003cspan additionalcitationids=\"CR37\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The nanoconjugates(Ab CFZ-LF-NPs) were repeatedly washed with Milli Q water followed by lyophilization using the optimized method. The nanoconjugates were collected after lyophilization and were subjected for ELISA examination to determine the percent of anti-Her2 conjugation and to measure the amount conjugated with CFZ-LF-NPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 FITC conjugation:\u003c/h2\u003e \u003cp\u003eFITC is prompt to bind with amino acids of antibodies, peptides, and proteins[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. Briefly, the CFZ-LF-NPs and AntiHer2-CFZ-LF-NPs in a molar ratio of 1:2 w/w, were dissolved in sodium carbonate/bi carbonate buffer solution (Na2CO3/NaHCO3, 25mM, pH 9.8) with substantial addition of FITC(0.1mg/ml). The resulting solution was allowed to stir with continuous agitation in incubator shaker at room temperature for 4 hours. The resulting fabricated nanoconjugates were centrifuged at 10,000 rpm, 4⁰C in a triplicate manner to remove excess unconjugated residues. The product was washed repeatedly to obtain FITC-CFZ-LF-NPs and FITC-AntiHer2-CFZ-LF-NPs.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Nanoparticles characterization and evaluation:\u003c/h2\u003e \u003cp\u003eParticle size distribution, polydispersity index (PDI) and zeta [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]potential of CFZ-LF-NPs and Anti-Her2-CFZ-LF-NPs. was evaluated using dynamic light scattering(DLS, Malvern Z90 zetasizer). Briefly, dispersion of nanoparticles(50mg) was prepared by reconstituting in water for injection(WFI) to prepare 5mg/ml concentration, further dispersing it in water for injection (WFI) to prepare a 5mg/ml, with simultaneous vortexing for 2 minutes at 2.5 vortex level in order to obtain a uniform protein nanoparticulate dispersion. The resulting dispersion of nanoparticles with 1.334 refractive index, was taken in disposable polystyrene cell cuvette and analysed for measurement run at 25\u0026ordm;C and scattering at 90\u0026ordm; angle. The hydrodynamic diameter of intensity resulted was discerned by z-avg value. Each experimental run was performed in triplicate and analysed at 25\u0026deg;C for demonstrating distribution pattern of nano particulates, and results were cumulated using the average of triplicate values of samples. Both CFZ-LF-NPs and Anti-Her2-CFZ-LF-NPs were subjected to 1HNMR analysis, which was recorded on Variant 400-MHZ spectrometer for confirming the successful configuration of CFZ-LF-NPs and conjugation of Anti-Her2(AB) with CFZ-LF-NPs. The samples containing freeze dried NPs and nanoconjugates were prepared in a Teflon-sleeved NMR tube(4.0mm o.d., 3.2 mm i.d.,Wilmad Glass) by concurrently dissolving protein-based nanostructures(5mg) in D2O(0.4 ml, 0.6%v/v). The resulting NMR sampling and characterization was handled at ambient temperature and pressure. NMR experiments were performed on 400 and 500 MHz using Tetramethylsilane(TMS) in 1HNMR for chemical shift reference[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTransmission emission microscopy(TEM) was used for surface morphological examination of lactoferrin nanoconjugates. TEM examination was performed using CM120 microscope. Subsequent dilutions of nanosuspension were prepared in double distilled water and a small quantity of suspension was deposited on a grid coated with polymer-based copper film. It was then allowed to settled for few mins.Excess liquid was scrapped off, and uranyl acetate was added to the sample followed by allowing it to dry at room temperature to form a thin film which was further examined at 70kv[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFurther, The FITC concentration post conjugation with Ab-CFZ-LF-NP was determined using fluorescence spectrophotometer. Fluorescence spectra of FITC labelled nanoconjugates were obtained on a Jasco spectrofluorometer using a 1cm path length cell containing the nanoconjugates at concentration ranging 5\u0026ndash;20 \u0026micro;g at room temperature(25⁰C)[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. The samples were prepared by diluting in PBS solution and the spectra were examined at excitation wavelength of 495nm. The emission spectra were recorded between 200\u0026ndash;800 nm.\u003c/p\u003e \u003cp\u003eThe %EE of carfilzomib was evaluated and determined by HPLC-based developed method. The Carfilzomib peaks were integrated at 210nm. For determination of %EE of CFZ-LF-NPs and Anti-Her2-CFZ-LF-NPs, the nanoparticles were weighed at equal concentration of 2mg/ml and centrifuged at 10,000 rpm for two cycles by repeatedly dispersing the pellets in acetonitrile in order to separate the unentrapped drug and further collected supernatant was analysed for entrapment efficiency after computing the chromatograms resulted by validated HPLC analysis[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Drug release from the synthesized nanoparticles was performed using the dialysis bag method (cut-off 12\u0026ndash;14 kDa). The %DR was evaluated at both physiological condition (pH 7.4) and the conditions prevalent in tumor (pH 5.5). Drug release was quantified by measuring the concentration of the released drug(CFZ) in the surrounding solution of at regular time intervals using a validated HPLC method of analysis at 210nm.\u003c/p\u003e \u003cp\u003e The stability of the optimal formulation in suspension was evaluated for 6 months at at 4\u0026ndash;8\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;/45%\u0026plusmn;5% RH ( Refrigerator, RF) and 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C/65\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH ( Room temperature; RT) as per ICH guidelines. Samples were evaluated, at 15-30days interval. The physiochemical change in protein nanoparticles was recorded periodically by evaluating %EE, size distribution, and physical appearance[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 ELISA test for examining anti-Her2 concentration in nanoconjugates (FITC-Anti-Her2-CFZ-LF-NPs):\u003c/h2\u003e \u003cp\u003eThe amount of anti-Her2 conjugated with CFZ-LF-NPs was determined using ELISA plate reader(BioRad, USA). The sample aliquots ranging 100-600ng/ml of Ab-CFZ-LF-NPs, were prepared in 0.1M PBS (pH7.4) and ELISA plate reading was employed using the developed protocol as follows; The plates were washed thrice after incubation with PBS (0.15M, pH7.2)containing 0.05% Tween 20(PBS-T) for 1.5 h at 37 ◦C. The plate was coated with recombinantly obtained extracellular matrix of antigen(0.5\u0026micro;g/ml of HER2 in PBS) and incubated overnight at 4⁰C. (0.5ug/ml). Blocking Buffer containing PBS with 3% non fat skimmed milk, was used to block the prepared ELISA plates, and kept in dark for 1.5hours at 37⁰C. Subsequently, Ab-CFZ-LF-NPs to the each well at different dilutions. Titrate the uncoupled antibody(1\u0026ndash;100ng/ml) substantially to construct as standard-curve. The plate was washed repeatedly and HRP-conjugate(secondary antibody) to each well containing different concentration of serial dilutions and incubate for 1.5 h. The colorimetry reaction was revealed using enzyme substrate,ie., 3,3,5,5-tetramethyl benzidine(TMB) and the optical density was measured at 450nm using ELISA plate reader[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 In vitro cytotoxicity of CFZ loaded nanoparticles.\u003c/h2\u003e \u003cp\u003e The in vitro cell cytotoxicity and co localization was perfomed on SKOV 3 cell, which was obtained from the American Type Culture Collection (ATCC), USA.\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 Cell cytotoxicity studies(MTT assay):\u003c/h2\u003e \u003cp\u003eThe MTT assay was performed to assess cell cytotoxic potential of CFZ-LF-NPs and FITC-Anti-Her2- CFZ-LF-NPs in SKOV-3(labelled cells for OC). Briefly, the cells at a density of 5 \u0026times;10\u003csup\u003e3\u003c/sup\u003e cells per ml. cells/well were seeded onto 96-well plates for overnight incubation at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e. After 24 h, the medium was replaced with 100 \u0026micro;L of varying concentrations of Free drug(CFZ), CFZ-LF-NPs, and FITC-Anti-Her2-CFZ-LF-NPs, ranging between 10-100ng/ml, in sterile media DMEM fresh. After 8 h the growth medium was changed by fresh medium to remove the non-internalized NPs and cultured for additional 24 h. Fed the plate with complete fresh media, and was then washed with PBS(pH 7.4) at the end of growth period, and 100\u0026micro;l of formazan solution(MTT, 1mg/ml) followed by filtering the resulting solution with 0.2 \u0026micro;m membrane filter. The plates were incubated at 37\u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e incubator for 4hrs. The media containing the untreated MTT was removed. Untreated cells in complete media(DMEM) were considered as negative(-)control. Triton X100 was further added in wells containing untreated cells, were considered as positive(+)control. The treatment throughout the assay was employed in triplicate Also, the cytotoxicity of NPs was evaluated in the HER2-negative ovarian cancer cell line A2780. The absorbance at 570 nm of each well was measured along-side a reference filter of 655nm using an ELISA-based microplate reader(BioRad,USA). The viable cells and %dead cell inhibition was cumulated average cell count method, wherein each absorbance of controls and samples represents the intensity of colorimetric reduction reaction of formazan take up by the treated and untreated cells, respectively. Statistical analysis of viability data was performed by 2-way ANOVA with Tukey\u0026rsquo;s multiple comparisons test.\u003c/p\u003e \u003cp\u003eIn order to determine the anti-tumor effect the IC50 of nanocarriers was also determined linear regression plot using cell Inhibition versus concentration of testing reagent[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2 Cellular uptake assay:\u003c/h2\u003e \u003cp\u003eCellular uptake of CFZ-LF-NPs, and FITC-Anti-Her2-CFZ-LF-NPs was analyzed by Confocal microscopy. Briefly, silicone supported coverslips (SecureSlip, Grace Bio-Labs) were placed in 6 well plates, then, 50,000 cells/well were cultured for 48 h with 2 mL of complete DMEM medium. The cells were then treated with free drug, as well as NPs (Free drug as control group, CFZ-LF NPs, and, FITC-AntiHer2-CFZ-LF-NPs) and Free FITC at fixed concentration determined after cytotoxicity analysis for growth period of 2 to 72 hours. Whereas, Her2 negative cell line was used as another control for the study. After this incubation period, medium was removed, and cells were washed twice with 500 \u0026micro;L of PBS to remove NPs that were not attached to or internalized by cells. The cells were collected for trypsinization. The trypsinized cells were neutralized by centrifuging at 1000rpm for 5 mins. The centrifuged cells were washed thrice with PBS(0.01M, pH7.4) to discard the debris or untreated dye. Cells were fixed in tissue culture dishes with 500 \u0026micro;L of 4% p-formaldehyde for 10 minutes, washed twice with PBS, and the coverslips were mounted on microscope slips with FluoroShield\u0026trade;/DAPI[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Slides were analyzed with an AX10 Zeiss Microscope, using DAPI and Alexa Fluor filter sets to visualize the nuclei and NPs, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3 Apoptosis and cell viability count via Flow cytometry:\u003c/h2\u003e \u003cp\u003eThe seeded cells(1\u0026times;10\u003csup\u003e5\u003c/sup\u003e )were stained with 10\u0026micro;l of Annexin V-FITC and PI for 10 mins at room temperature by incubating in the dark according to protocol of biosciences. The viable cell count and apoptotic cells were quantified using flow cytometer(Attune nxt, Thermofischer Scientific). The cells were quantified and observed in CellQuest solftware and the results are expressed as rate and percentage of apoptosis(late and early apoptosis)[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.8 In vivo anti-tumor efficacy studies:\u003c/h2\u003e \u003cp\u003e \u003cstrong\u003e\u003cb\u003eEthical a\u003c/b\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003epp\u003c/span\u003e\u003cb\u003eroval\u003c/b\u003e\u003c/strong\u003e \u003cp\u003e \u003cb\u003eand accordance\u003c/b\u003e \u003c/p\u003e \u003c/p\u003e \u003cp\u003eAll \u003cem\u003ein vivo\u003c/em\u003e experiments on animal were approved by the Institutional Animal Care and Use Committee of \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eIAEC Approval No\u003c/span\u003e: IAEC/PIPH/09/23. Moreover, these experiments were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)and ARRIVE 2.0 guidelines to ensure transparency and reproducibility (Animal Research: Reporting of \u003cem\u003eIn Vivo\u003c/em\u003e Experiments).\u003c/p\u003e \u003cp\u003eMoreover, the experimental animals were procured from \u003cb\u003eSun Pharmaceutical Industries Ltd.\u003c/b\u003e, India, a licensed commercial supplier of laboratory animals(Vadodara, Gujarat) following approval of the experimental protocol from Institutional Animal Ethics Committee (IAEC)\u003c/p\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.8.1 Tumor distribution studies:\u003c/h2\u003e \u003cp\u003eThe in vivo anti tumor effect was studies and tumor distribution studies were conducted based on the above mentioned protocol approval. Twenty healthy female athymic mice(6\u0026ndash;8 weeks) old were commercially obtained from \u003cb\u003eSun Pharmaceutical Industries Ltd.\u003c/b\u003e, India, a licensed commercial supplier of laboratory animals(Vadodara, Gujarat) and housed in pathogen free environment with access to food and water. The mice were injected intraperitoneally with 1\u0026times;10\u003csup\u003e7\u003c/sup\u003e viable SKOV-3 cells to establish tumor bearing models. After 15 days of growth, the mice were divided into three random groups of five mice: Group 1- PBS (saline control), Group 2: CFZ solution, Group 3: CFZ-LF nanocarriers. For determining tumor distribution, priorly the standard curve of CFZ in blood, and homogenized tissue samples(liver, spleen, kidney, heart, lung, uterus, small intestine, and tumor tissues) of tumor bearing mice was performed 20 \u0026micro;l different quantitative standard CFZ solutions (0.95, 1.9, 9.5, 19, 38, 76, and 152 \u0026micro;g/ml) that was added to homogenate samples (200 \u0026micro;l) using HPLC analysis[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The linear regression equation was then established based on the CFZ concentration (X) and AUC (Y) value from the HPLC test. Each mice of respective group was injected intraperitoneally with either CFZ or CFZ-LF nanocarriers with CFZ concentration of 5 mg/kg alone or in nanoparticles. Mice were anesthetized using [e.g., isoflurane / ketamine\u0026ndash;xylazine] prior to sacrifice. At predetermined time points (1, 2, 4, 6, 12, and 24 h), 5 mice in each group were euthanized. Euthanasia was performed in accordance with CPCSEA guidelines using [method, e.g., CO₂ asphyxiation]. Further, their plasma and tissue samples were quickly collected for an HPLC test and then calculated based on the linear regression equation obtained as mentioned above to determine the CFZ concentration in the samples[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Based on concentration of drug reached varied organs was quantified using HPLC based standard curve, the tumour distribution in separate homogenized organs was determined.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.8.2 Tumor Growth inhibition study(anti-tumor effect):\u003c/h2\u003e \u003cp\u003eThe tumor bearing mice were randomly divided in four groups(6/group): Group 1: PBS(control), Group 2: free CFZ, Group 3: FITC-CFZ-LF-NPs, and Group 4: FITC-Anti-Her2-CFZ-LF-NPs. A single dose of CFZ alone and in nanocarriers was intravenously administered to each mouse and saline solution was used as a control. Each mouse received a treatment of 120 \u0026micro;L via intraperitoneal injection once a week for two weeks. All samples were freshly prepared and filtered prior to injection[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. The mice were inspected daily and the overall body condition and body weight were measured. The tumor sizes and animal body weights were then measured every 3 days. After thorough observation, at the endpoint of treatment, the mice were euthanized and organs(liver, spleen, kidney, heart, lung, uterus, small intestine, and tumor tissues) were collected and placed in 10% formalin for Hemotaxylin \u0026amp; Eosin (H\u0026amp;E) followed by immunohistochemistry was performed. Moreover, the average tumor volume was calculated that was used to determine the effectiveness of treatment.\u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical analysis\u003c/b\u003e:\u003c/p\u003e \u003cp\u003eThe results were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Student\u0026rsquo;s t-test in Excel was used to analyse the statistical significance of the tumour growth inhibition. A value of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Result and discussion","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Nanoparticles characterization and evaluation:\u003c/h2\u003e \u003cp\u003e\u0026lsquo;Carfilzomib loaded lactoferrin nanoparticles\u0026rsquo; (CFZ-LF-NPs), and anti-Her2 nanoconjugates(Anti-Her2-CFZ-LF-NPs) were ascertained for their particle size and zeta potential as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea \u003cb\u003eand b\u003c/b\u003e, by using dynamic light scattering (DLS) in Malvern Z90 zetasizer equipped with internal laser. The particle size distribution, PDI, and zeta potential was found to be in the range of 80 to 250 nm, 0.2 to 0.7 and +\u0026thinsp;2.01 to +\u0026thinsp;10.45 for CFZ-LF-NPs that indicates the heterogeneously distributed polydisperse nanoparticulate system. Figure () delineating the particle size pattern of distribution and zeta potential of optimal composition of CFZ-LF-NPs, which was found to be 80.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.014 nm and +\u0026thinsp;14.58 zeta potential(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). This indicates the narrow uniform size distribution with 0.227\u0026thinsp;\u0026plusmn;\u0026thinsp;0.032 PDI as well as net positive surface charge indicates that lactoferrin is positively charges below isoelectric point, delineating suitable stability that was anticipated for lactoferrin (with pI 8 )[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. However, after conjugating with Anti-Her, the particle size of Anti-Her2- CFZ-LF-NPs was found to be 240\u0026thinsp;\u0026plusmn;\u0026thinsp;0.224nm and 0.357\u0026thinsp;\u0026plusmn;\u0026thinsp;0.412PDI as well as zeta potential was observed to be 26.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.147(Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb\u003cb\u003e)\u003c/b\u003e, indicating the potential stability of formulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e\u0026lsquo;Carfilzomib loaded lactoferrin nanoparticles\u0026rsquo; (CFZ-LF-NPs) synthesize by Desolvation, subjected \u003csup\u003e1\u003c/sup\u003eH NMR spectroscopy The Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, b, c, d, showing the NMR spectra of, CFZ- LF-NPs, free EDC NHS, AntiHer2-CFZ-LF-NPs before and after dialysis to confirm the removal of unreacted residues. The multiple signals observed between 3.2 to 4.8 ppm were assigned to the resonance of methylene protons -Ch3-, depicting the evident spectrum peaks corresponding to LF. In addition. signals at 2.78 ppm assigned to aliphatic protons -CH2-NH-C\u0026thinsp;=\u0026thinsp;O linked secondary amine corresponding to peptide chain of LF. Aromatic amine, and peaks of deshielded aromatic proton at downfield between, as observed in free LF, which can be also seen in NMR spectra of CFZ-LF-NPs. The NMR spectrum were acquired as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, showing the conjugation of AntiHer2 and CFZ-LF-NPs. The NMR signals in CFZ-LF-NPs, showing the chemical shift of methylene protons at up-field can be evidently observed in Ab-CFZ-LF-NPs at 4.96 ppm(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The shift may be due to electronegativity of neighbouring groups belonging to Anti-Her2. Moreover, antibodies are much high in molecular weight, so clusters of signals may be observed that may results in noise interpretation. However, the small signals at 5.2 ppm, attributes to CONH formation, confirming the conjugation(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec \u003cb\u003eand d\u003c/b\u003e). Also, compared with free EDC- NHS(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb) showing signals in methylene regions, while no signals were acquired in AntiHer2-CFZ-LF-NPs(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). The removal of isourea(byproduct of EDC-NHS reaction) was confirmed by NMR of Ab conjugates before and after dialysis(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec \u003cb\u003eand d\u003c/b\u003e). The signal observed between 2.5 to 3 attributes to methyl group of isourea byproduct\u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb \u003cb\u003eand c\u003c/b\u003e), as shown in NMR of AntiHer2-CFZ-LF-NPs(before dialysis), and the intensity of which is completely reduced after dialysis as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). Moreover, the intensity of signals of succinimide ring attributed at 2.78 ppm were completely reduced after dialysis in AntiHer2-CFZ-LF NPs, as compared to evident peaks in NHS(Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, c \u003cb\u003eand d\u003c/b\u003e) with high intensity. This confirmed the removal of byproducts and unreacted EDC-NHS and its intermediate product[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe antibody conjugation labelled with FITC dye can be emerged as cancer theragnostic tool for biological imaging. Moreover, FITC have the capability to react with amine reactive groups on proteins and antibody inside and outside the body, hence FITC(0.1mg/ml) was used for conjugated with Anti-Her2-CFZ-LF-NPs. The fluorescence spectrophotometer(Jasco) was used for determining the FITC content in nanoconjugates to demonstrate a fluorophore intensity for biological imaging purpose[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. The substantial rise in fluorescence intensity(FI) with concurrently augmenting concentration of nanoconjugates, encompasses the FITC conjugation of nanoconjugates was compared with Free FITC as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e \u003cb\u003e).\u003c/b\u003eThe experiments were conducted in triplicate and the concentration of FITC in nanoconjugates was calculated using the average absorbance, which resulted in 18.14 \u0026micro;g/ml and 19.04 \u0026micro;g/ml concentration in FITC-AntiHer2-CFZ-LF-NPs, thereby confirming more than 90% conjugation with R-square values,i.e., r2\u0026thinsp;=\u0026thinsp;0.9901(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Therefore, high linearity near to 1 entail less than 2%RSD with high FITC conjugation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSurface morphological examination of protein nanoconjugates(CFZ-LF-NPs and FITC-Anti-Her2-CFZ-LF NPs) was assessed by Transmission emission scanning microscopy as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea \u003cb\u003eand b\u003c/b\u003e, which demonstrate the spherical shape of lactoferrin nanoparticulate system uniformly distributed within size range of 80 to 200 nm.\u003c/p\u003e \u003cp\u003eThe entrapment efficiency of CFZ was calculated by reconstituting the freeze-dried product as per the above-mentioned procedure and centrifuged at 10,000 rpm, where methanol or acetonitrile was used as a diluent to separate the un-entrapped drug in the supernatant, which was further analysed for drug entrapment by HPLC[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. The entrapment efficiency of drug in CFZ-LF-NPs was found to be 86% \u0026plusmn; 0.289 with %DL of 14.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.521%. This was determined by using a validated HPLC method for carfilzomib estimation at 210 nm wavelength, as shown in \u003cb\u003esupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. Sharp chromatogram of isolated supernatant samples containing free drug separated at 7.45 RT in HPLC( \u003cb\u003esupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). However, disappearance of CFZ peak with HPLC chromatogram of LF (11.2 min RT) having sharp resolution shown in \u003cb\u003esupplementary Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003econfirmed the drug entrapment.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe CFZ-LF-NPs were subjected to in vitro drug release using Franz diffusion cell, at physiological condition (pH 7.4) and the conditions prevalent in tumor (pH 5.5). The \u003cem\u003ein vitro\u003c/em\u003e drug release curve is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. \u003cem\u003eIn vitro\u003c/em\u003e drug release was investigated by developed and validated HPLC method. The results obtained from drug diffusion study of CFZ-LF-NPs at pH 7.4 was found to be 65.258\u0026thinsp;\u0026plusmn;\u0026thinsp;0.382% (up to 8 days) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. It could be concluded that the invented formulation was able to prolong the drug release continuously up to a week avoiding frequent application of the formulation. The final formulation of CFZ-LF-NPs had shown fast and highest drug release of 91.585\u0026thinsp;\u0026plusmn;\u0026thinsp;0.017% (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) at tumor pH of 5.5 up to 96 hours, and sustained the drug diffusion at physiological condition till 8 days. This observation delineates pH sensitive drug release by the formulation (CFZ-LF-NPs) that was one of the objectives to prolong the retention of the active drug at physiological pH and enhance the diffusion at tumor microenvironment (pH 5.5). Whereas, untreated drug was released shortly within 72 hrs. This concluded that the drug conjugate of protein nanoparticles is capable of retaining the drug physiologically and sustained the diffusion with pH sensitive response at acidic pH (5.5). The in vitro drug release was quantified by developed and validated HPLC method for estimation of carfilzomib at 210 nm as shown in \u003cb\u003esupplementary Figure S2\u003c/b\u003e, with chromatogram taken at variable time in hours.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe long term stability study was employed to evaluate and observe the changes in the quality, safety, appearance, compatibility of the formulation when exposed to various temperature conditions and to observe how the parameters keep on changing over the time of storage. Stability studies revealed that the nanoparticles stored over a storage of 6 months when kept at both refrigerated temperature as well as room temperature, whereas storage at 4\u0026ndash;8\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;/45%\u0026plusmn;5% RH resulted with minimal changes in size and %EE as compared to 25\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C/65\u0026thinsp;\u0026plusmn;\u0026thinsp;5% RH, hence this delineates the stability of protein nanoparticles more under refrigerated conditions (\u003cb\u003esupplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e ). Thus, purpose is to obtain a long-term storage of the formulation to maintain the shelf life the formulation was successfully employed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.2 ELISA A test for examining anti-Her2 concentration in nanoconjugates (FITC-Anti-Her2-CFZ-LF-NPs):\u003c/h2\u003e \u003cp\u003eELISA results(\u003cb\u003esupplementary Figure S3\u003c/b\u003e) revealed that successful conjugation of Anti-Her2 with rise in optical density at higher concentration of FITC-Anti-Her2 conjugated Lactoferrin. Nanoparticles. Highest optical density achieved 600 ng/ml of FITC-Anti-Her2 conjugated Lactoferrin Nanoparticles indicates irrefutable in its targeting affinity but also confirms successfully employed conjugation. Substrate TMB oxidizes to TMB\u0026thinsp;+\u0026thinsp;2 when it reacts with HRP-conjugate and after adding the stop solution (acid) the colour changes from blue to yellow due to HRP degradation and acidification of TMB. The entire enzymatic reaction reveals estimation of target protein. The intensity of colour indicates the amount of antibody conjugated with target antigen via enzyme substrate affinity towards the primary and secondary antibody. The optical density inclined with increasing concentration that can be inferred from \u003cb\u003esupplementary Figure S3\u003c/b\u003e. The optical density was found to be within limits when compared with standard. However more than 60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.225% of AB conjugation was recovered, depicting the coupling of anti-Her(1 mg/ml) with CFZ-LF-NPs [52,55,56].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.3 In vitro cytotoxicity of CFZ loaded nanoparticles.\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003e3.3.1 Cell cytotoxicity studies(MTT assay):\u003c/h2\u003e \u003cp\u003eThe cell culture examination was erformed for determination the percentage cell inhibition(MTT assay) of nanoformulation in labelled SKOV-3 cells, including CFZ-LF-NPs, and FITC-AntiHer2-CFZ-LF-NPs and Carfilzomib alone(control)[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The MTT assay for determining the cell cytotoxicity was assessed till 72 hours of treatment. It was observed that Anti-Her2 nanoconjugates(FITC-AntiHer2-CFZ-LF-NPs) showed more than 90% of cell inhibition,i.e., 95.72\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44% at 5nM concentration which is 2-fold enhanced inhibition as compared to free drug. Whereas, CFZ-LF-NPs shown 66.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 % cytooxicity that is less than FITC-AntiHer2-CFZ-LF- NPs. Unlikely, plain drug resulted in least cell cytotoxicity with 45.60\u0026thinsp;\u0026plusmn;\u0026thinsp;2.15% even at the highest concentration of 25nM as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e. Therefore, the results clearly unravels high efficacy of Anti-Her2 nanoconjugates(FITC-AntiHer2-CFZ-LF-NPs), as antibody conjugation serves as dual therapeutics against cancer with target specific uptake, as compared to CFZ-LF-NPs (non-conjugated) 60% cell inhibition thereby depicting target specificity for inducing cell death. Hence, the nanoconjugates can be emerged as a futuristic nano-cargo for cancer theragnostic in molecular imaging.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, anti-tumor effect was evaluated \u003cem\u003ein vitro\u003c/em\u003e by determining IC\u003csub\u003e50\u003c/sub\u003e values. The nanoparticles and nanoconjugates resulted with 11.167nM(AntiHer2-CFZ-LF-NPs), and 13.447nM(CFZ-LF-NPs) of IC50, thereby exhibiting a 2-fold reduction in inhibitory concentration of drug in nanoconjugates as compared to Carfilzomib alone as single drug therapeutics with 25.231nM IC50. Thereby, indicating nanoformulation enhanced therapeutics of drug with minimum bare concentration for cell inhibition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section3\"\u003e \u003ch2\u003e3.3.2 Cellular uptake assay:\u003c/h2\u003e \u003cp\u003eThe cellular uptake was ascertained using dye staining under confocal microscopy to determine the cell internalization and apoptosis of CFZ-LF-NPs and FITC-Anti-Her2-CFZ-LF-NPs that was compared with non treated cells(PBS) as negative control and free CFZ as positive control. The live cells were stained using DAPI and dead cells were stained with propidium iodide using Annexin-V kit[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. The results revealed highest cellular uptake of FITC-Anti-Her2-CFZ-LF-NPs as compared to CFZ-LF-NPs in Her2 labelled SKOV-3 cells due to enhanced target specificity of Anti-Her2 conjugated lactoferrin nanoparticles as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b, c, d. Moreover, iron de-efficiency is highly overexpressed in proliferating cells, thereby iron containing lactoferrin synergistically contributed in cell internalization. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ed unravels tumor necrosis was observed in Anti-Her2 treated cells after 72 hours, with red chromatin nuclei (PI stained cell death). The red stained chromatin fibrosis in cells is high observed in FITC-AntiHer2 -CFZ-LF-NPs as compared to CFZ LF-NPs. Hence, LF NPs highly taken up via iron de-efficient cancer cells, whereas Anti-Her2 conjugation prominently shown high stained dead cells as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ec \u003cb\u003eand d\u003c/b\u003e. Therefore, the study encompasses the dual cancer therapeutics of using Anti-Her2 with carfilzomib loaded LF NPs with target specificity against OC cancer phenotypes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section3\"\u003e \u003ch2\u003e3.3.3 Apoptosis and cell viability count via Flow cytometry:\u003c/h2\u003e \u003cp\u003eThe SKOV-3 cells were treated with CFZ(5nM), CFZ-LF-NPs(5nM), and FITC-AntiHer2-CFZ-LF-NPs(nM) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, b, c) followed by 24 cell culturing and staining with Annexin-V and PI(propidium iodide) kit for determining the apoptosis in cell population in different time intervals. The Annexin-V having the ability to interact with phosphatidylserine of a live cell with initial stage of apoptosis and PI binds penetrates in dead nuclei of cells for labelling dead cells. However, the apoptotic cell population can be identified by separate particles in black dot diagram as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e with flow cytometry results at different time intervals(48 and 72 hours), in which there are 4 quadrants. Amongst the four quadrants, the lower left quadrant(Q3) depicts viable cells,i.e., the cells which are negative for PI and Annexin uptake, while the cells which are positive for Annexin-V and negative for PI are demonstrated as early apoptotic cells as distributed in lower right quadrant(Q4). In this early apoptosis, the cell membrane begin to shrink resulting in increasing intensity of side scatter in flow cytometer. The cells having positive staining of both PI and Annexin-V are late apoptosis/necrotic cells shown in upper right quadrant(Q2), while the cells which are PI positive and AnnexinV-negative are necrotic cells shown in upper left quadrant(Q1). This can be inferred from figure, depicting the clustered of cell population in control, that reduced and the separate cell entity increased in CFZ-LF-NPs, FITC-AntiHer2-CFZ-LF-NPs, thereby showed increase in apoptosis percentage with cell shrinkage. The apoptosis percentage of CFZ, CFZ-LF-NPs, and FITC-AntiHer2-CFZ-LF-NPs was observed to be increased from 9%, 27%, and 39% to 23%, 64.5%, and 70.8% in 72 hours of treatment. This increment from 24 to 72 hours treatment, demonstrate a timely-dependent apoptosis. Noticeably, CFZ alone shown incremental rise in apoptosis in initial 12 hours after which a steep decline in rise was observed Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea. As anticipated, FITC-AntiHer2- CFZ-LF-NPs showed the highest late apoptosis compared to CFZ-LF-NPs as observed in Q2(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb \u003cb\u003eand c\u003c/b\u003e), and cell populations in Q1, thereby depicting a necrosis followed by late apoptosis indicating high cell death. However, a shift from high intensity of side scatter to front scatter was observed in FITC-AntiHer2-CFZ-LF-NPs(Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). As reduced side scatter(SSC) and increased(FSC) determines a complete cell shrinkage resulting late apoptosis/necrosis. Moreover, FITC-AntiHer2-CFZ-LF-NPs showing completely inclined FSC, thereby demonstrating high apoptosis due to dual synergistic effect of anti-Her2 and CFZ in SKOV-3 cell population. Moreover, the increase in apoptosis percentage induced in cells treated with CFZ-LF-NPs, FITC-AntiHer2-CFZ-LF-NPs, encompasses an enhanced drug efficacy through aid of protein nanoconjugates[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.4 In vivo anti-tumor efficacy studies:\u003c/h2\u003e \u003cdiv id=\"Sec24\" class=\"Section3\"\u003e \u003ch2\u003e3.4.1 Tumor distribution studies:\u003c/h2\u003e \u003cp\u003eThe CFZ levels of plasma and other tissues were studied at different points delivered from conjugated(CFZ-LF-NPs and non-conjugated(FITC-AntiHer2-CFZ-LF-NPs nanoparticles as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. Firstly, peak extended time delineating absorbed concentration in all nano carrier groups were compared with the free drug(CFZ) in different areas of tissues including tumor tissues as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. These results again showed slow drug release in nanocarrier groups with gradual rise in concentration in tissues, whereas initial controlled release effect. While, free drug concentration was high initially, that showed steep decline with less penetration to deep tissues(Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Most importantly in tumor tissues, the CFZ concentration in FITC-AntiHer2-CFZ-LF-NPs(Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e) at 12 hours were much higher than nonconjugated CFZ-LF-NPs and free CFZ as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e, while there was a negligible statistical difference between the latter two groups in other tissues. These results in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e clearly delineate the AntiHer2 and LF mediated active targeting efficacy of FITC-AntiHer2-CFZ-LF-Nps at target tumor site with high penetration and uptake as compared to normal tissues[57]. This indicates less plausible dose dependent toxicity in normal tissues with nanoconjugates. However, except for liver, uterus, and spleen CFZ-LF-NPs has greater uptake in other tissues, that delineated not target specificity. Moreover, in Heart, Kidney, and lung significantly low levels of CFZ in FITC-AntiHer2-CFZ-LF-NPs was observed. This indicates improved pharmacological effect of lactoferrin nanoconjugates of carfilzomib by preventing drug accumulation in non-target tissues, thereby reducing the toxicity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003e3.4.2 Tumor Growth inhibition study:\u003c/h2\u003e \u003cp\u003eThe final tumor size in treated mice were notably reduced in FITC-AntiHer2-CFZ-LF-NPs as observed in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e, while there was less statistical difference between free CFZ and CFZ-LF-NPs groups(p\u0026thinsp;\u0026lt;\u0026thinsp;0.005). The final tumor size in FITC-AntiHer2-CFZ-LF-NPs treated mice was found to be 312.67\u0026thinsp;\u0026plusmn;\u0026thinsp;41.45 mm\u003csup\u003e3\u003c/sup\u003e), much smaller than those treated with CFZ-LF-NPs(487.13\u0026thinsp;\u0026plusmn;\u0026thinsp;32.12 mm\u003csup\u003e3\u003c/sup\u003e) and free CFZ(508.56\u0026thinsp;\u0026plusmn;\u0026thinsp;39.33 mm\u003csup\u003e3\u003c/sup\u003e) groups as well as controls (805.49\u0026thinsp;\u0026plusmn;\u0026thinsp;55.23 mm\u003csup\u003e3\u003c/sup\u003e) as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e. All the treatment groups exhibited significant antitumor effect as compared to the control groups(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05)[57]. The tumor growth curve showed that FITC-AntiHer2-CFZ-LF-NPs has much stronger antitumor effect as compared to other groups of CFZ-LF-NPs and free CFZ (Fig ). The inhibitory rate based on tumor volume were 61.88% in FITC-AntiHer2-CFZ-LF-NPs, that is 2-fold times higher than in CFZ-LF-NPs(27.23%) and free CFZ(23.12%). Negligible changes in body weight in all the four groups(p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) indicated no obvious side effects in the nano-carriers groups.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe research work emphasizes surface functionalized carfilzomib loaded lactoferrin nanoparticles using different biomarkers for molecular recognition such as anti-Her2(Herceptin). CFZ was chosen for several reason concerned with its potential clinical outcomes, irreversible action, and motif to prevent its peptide mediated clearance and enhance its prolongation. However, protein are more susceptible of protein corona formation with serum proteins body. Therefore, to prevent this rigidizing its surface with molecular tag can be potential approach. The lactoferrin nanoparticles were functionalized with anti-Her2 using EDC and NHS based carbodiimide chemistry. The above discussed formulations synthesized and were evaluated for anti-tumor activity in SKOV-3 cells for determining the cell cytotoxicity, cellular uptake, apoptosis mechanism, and viable cell count using plate reader, confocal imaging, and flow cytometry. The results of these studies revealed 2 fold times tumor inhibition in mice treated with nanoconjugates(FITC-AntiHer2-CFZ-LF-NPs) with same dose that of free CFZ and CFZ-LF-NPs(5 mg/kg), underscoring potential target specific treatment. This antitumor effect in such metronomic dose can be a promising approach in cancer treatment. There was no reccurence was observed in treated mice indicating improved survival \u003cem\u003ein vivo\u003c/em\u003e. The prominent results and observation indicates beyond the scope, that the developed biomarkers tagged carfilzomib loaded lactoferrin nanoparticles may emerge as spy-catching tool in combination with therapeutics for dual targeting with enhanced stability for intravenous administration in cancer theranostics.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics Approval and Consent to Participate\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical\u0026nbsp;\u003c/strong\u003eapproval and\u0026nbsp;\u003cstrong\u003eaccordance\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHuman and Animal Rights\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo human participants were involved in this study.\u0026nbsp;All \u003cem\u003ein vivo\u003c/em\u003e experiments on animal were approved by the Institutional Animal Care and Use Committee of \u003cu\u003eIAEC Approval No:\u0026nbsp;\u003c/u\u003eIAEC/PIPH/09/23. Moreover, these experiments were conducted in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA)and ARRIVE 2.0 guidelines to ensure transparency and reproducibility (Animal Research: Reporting of \u003cem\u003eIn Vivo\u003c/em\u003e Experiments).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental animals(athymic mice)\u0026nbsp;were procured from \u003cstrong\u003eSun Pharmaceutical Industries Ltd.\u003c/strong\u003e, India, a licensed commercial supplier of laboratory animals(Vadodara, Gujarat) following approval of the experimental protocol from Institutional Animal Ethics Committee (IAEC).\u003c/p\u003e\n\u003cp\u003eAs the animals were obtained commercially from an authorized supplier, no additional institutional permission or owner consent was required beyond approval from the Institutional Animal Ethics Committee (IAEC). The relevant details have been updated in the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for Publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials: This contribution is not included with any extra files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe author would like to acknowledge the financial assistance from Gujarat State Biotechnology Mission (GsBTM), Gandhinagar for project work.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eQu H, Yang J, Li S, Xu J, Zhou X, Xue X, et al. Programmed-response cross-linked nanocarrier for multidrug-resistant ovarian cancer treatment. 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Journal of Experimental and Clinical Cancer Research. 2018;37(1). https://doi.org/10.1186/s13046-018-0700-z \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Biofunctionalization, Lactoferrin, self-assembly, Anti-Her2, Cellular imaging","lastPublishedDoi":"10.21203/rs.3.rs-8494829/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8494829/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eThe study aimed fabricate Anti-Her2 carfilzomib loaded lactoferrin nanoparticles in order to address the shortcomings of carfilzomib related to poor half life, low solubility, and poor \u003cem\u003ein vivo\u003c/em\u003e stability. The objective was to modify the programmable assembly of protein with biomarkers and imaging probe to ameliorate precise targeted theragnostics against ovarian cancer\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e \u003cp\u003eCarfilzomib loaded lactoferrin nanoparticles were fabricated using Desolvation technique to obtain optimal composition using Full factorial design. Further, surface functionalization with FITC tagged anti-Her2(Ab) was performed using EDC-NHS carbodiimide chemistry. Detailed characterization and in vitro evaluation including cellular uptake studies in SKOV-3 cells were performed.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe carfilzomib loaded lactoferrin nanoparticles(CFZ-LF-NPs) were found to be uniformly distributed in range of 100-150nm. The percentage drug loading and entrapment efficiency was found to be 14.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.521% and 86% \u0026plusmn; 0.289, respectively. CFZ-LF-NPs showed prolong drug release till 8 days at physiological pH(7.4), while 91.932\u0026thinsp;\u0026plusmn;\u0026thinsp;0.549% of drug release at tumor pH 5.5 on day 4, thereby indicating pH triggered drug release. NMR, and fluorescent microscopy confirms successful conjugation of anti-Her2(Ab) with CFZ-LF-NPs. ELISA test for anti-HER2 conjugated NPs(FITC-AntiHer2-CFZ-LF-NPs) revealed the highest percent of antibody(\u0026gt;\u0026thinsp;60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.225%) conjugation with nanoparticles above the concentration of 5 ug/ml.The MTT assay, cellular uptake assay and tumor growth inhibition studies in treated mice unravels high cytotoxicity in nanoconjugates(FITC-AntiHer2-CFZ-LF-NPs) as compared to non-conjugated NPs and plain drug.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThe potential approach may emerge in future as a molecular-docks comprising a immunotherapy-based theragnostic in cancer.\u003c/p\u003e","manuscriptTitle":"Immunomodulatory and Antitumor Activity of Surface-Functionalized Lactoferrin Nanoparticles in Ovarian Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-09 16:43:05","doi":"10.21203/rs.3.rs-8494829/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-07T20:47:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T20:45:19+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-10T23:18:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T16:32:56+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T09:35:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-09T03:52:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288643000631432568731811635001256740041","date":"2026-04-05T05:07:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"166413508089343781767879628702157374449","date":"2026-04-03T19:31:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"299766235669890454937200861041845595856","date":"2026-04-03T12:03:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"311573341260782314238120924845901702019","date":"2026-04-03T09:12:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"103036627547007310628959158666263564532","date":"2026-04-03T06:26:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"46091166894732493320781533183296507472","date":"2026-04-03T06:14:55+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"88310670955493185385690735207714625875","date":"2026-02-23T13:54:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11528861099813648320008692252689492879","date":"2026-02-07T06:24:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-05T02:02:36+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-27T18:12:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-22T07:28:32+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-21T08:58:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Nano","date":"2026-01-21T08:14:47+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-nano","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"narl","sideBox":"Learn more about [Discover Nano](https://www.springer.com/journal/11671)","snPcode":"11671","submissionUrl":"https://submission.nature.com/new-submission/11671/3","title":"Discover Nano","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"80bbc3a6-88fc-432d-a447-b36e7c1cfea4","owner":[],"postedDate":"February 9th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-07T20:47:34+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-07T20:45:19+00:00","index":71,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T20:54:36+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-09 16:43:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8494829","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8494829","identity":"rs-8494829","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}