Recent advances in enzymatic biosensors for point-of-care detection of biomolecules.

Late state-of-the-art analytical methodologies in chromatography, spectroscopy, and electroanalysis have been developed to meet the challenges of changing environmental and health issues. The modern trends in developing new protocols emphasize economic, portable, nano, or even smaller sample sizes and less time-consuming processes. This has led to the development of technology-based biosensors which meet most of the above requirements. The lab-on-chip technology exploiting enzyme-based biosensors has made the analytical processes very efficient, accurate, affordable, and requiring nano-scale sample sizes. In this review, an attempt is being made to review the literature based on state-of-the-art technology of enzyme-based biosensors for the detection of biomolecules.

Printed oxygen gas sensor using copper-DTDTPA solid electrolyte.

We present a new method for the rapid and cost-effective fabrication of solid electrolyte-based printed potentiometric oxygen sensors working at ambient temperature using Cu-dithiolated diethylene triamine pentaacetic acid complex molecules (Cu-DTDTPA) adsorbed on Grade-1 laboratory filter paper and subsequent 3-D printing of interdigitated electrodes employing silver/silver chloride ink. The decrease in conductivity with time and frequency-dependent impedance response confirms the filter paper adsorbed Cu-DTDTPA as a solid electrolyte. A plausible structure of the Cu-DTDTPA solid electrolyte and its mechanism of reaction with oxygen are presented. A maximum sensitivity of 0.052 mV per %O2, the maximum response time of 1.15 s per %O2, a wide measurement output ranging from 14.55 mV to 17.25 mV for 20%-90% of O2 concentration, a maximum standard deviation of 0.12 mV in output voltage, almost similar trends of the response on temperature, humidity variations and ageing and high selectivity establish the sensor for use in medical ventilator applications, specifically in the COVID19 pandemic.

A paper based microfluidic device for easy detection of uric acid using positively charged gold nanoparticles.

A paper based microfluidic device is fabricated that can rapidly detect very low concentrations of uric acid (UA) using 3,5,3',5'-tetramethyl benzidine (TMB), H2O2 and positively charged gold nanoparticles ((+)AuNPs). In the presence of (+)AuNPs, H2O2 reacts with TMB to produce a bluish-green colour which becomes colourless on reaction with UA. This colorimetric method can detect as low as 8.1 ppm of UA within <20 minutes on white filter paper. This technique provides an alternative way for UA detection.

Laminin receptor specific therapeutic gold nanoparticles (198AuNP-EGCg) show efficacy in treating prostate cancer.

Systemic delivery of therapeutic agents to solid tumors is hindered by vascular and interstitial barriers. We hypothesized that prostate tumor specific epigallocatechin-gallate (EGCg) functionalized radioactive gold nanoparticles, when delivered intratumorally (IT), would circumvent transport barriers, resulting in targeted delivery of therapeutic payloads. The results described herein support our hypothesis. We report the development of inherently therapeutic gold nanoparticles derived from the Au-198 isotope; the range of the (198)Au ß-particle (approximately 11 mm in tissue or approximately 1100 cell diameters) is sufficiently long to provide cross-fire effects of a radiation dose delivered to cells within the prostate gland and short enough to minimize the radiation dose to critical tissues near the periphery of the capsule. The formulation of biocompatible (198)AuNPs utilizes the redox chemistry of prostate tumor specific phytochemical EGCg as it converts gold salt into gold nanoparticles and also selectively binds with excellent affinity to Laminin67R receptors, which are over expressed in prostate tumor cells. Pharmacokinetic studies in PC-3 xenograft SCID mice showed approximately 72% retention of (198)AuNP-EGCg in tumors 24 h after intratumoral administration. Therapeutic studies showed 80% reduction of tumor volumes after 28 d demonstrating significant inhibition of tumor growth compared to controls. This innovative nanotechnological approach serves as a basis for designing biocompatible target specific antineoplastic agents. This novel intratumorally injectable (198)AuNP-EGCg nanotherapeutic agent may provide significant advances in oncology for use as an effective treatment for prostate and other solid tumors.

Bombesin peptide conjugated gold nanocages internalize via clathrin mediated endocytosis.

The nature of interaction and mechanism of internalization of receptor-avid peptide nanoparticles with cells is not yet completely understood. This article describes the cellular internalization mechanism and intracellular trafficking of peptide conjugated receptor targeted porous Gold nanocages (AuNCs) in cancer cells. We synthesized and characterized a library of AuNCs conjugated with bombesin (BBN) peptide. Evidence of selective affinity of AuNC-BBN toward gastrin releasing peptide receptors (GRPR) was obtained using radiolabeled competitive cell binding assay. Endocytic mechanism was investigated using cell inhibitor studies and monitored using optical and transmission electron microscopy (TEM). Results show AuNC-BBN uptake in PC3 cells is mediated by clathrin mediated endocytosis (CME). Indeed, in the presence of CME inhibitors, AuNC-BBN uptake in cells is reduced up to 84%. TEM images further confirm CME characteristic clathrin coated pits and lysosomal release of AuNCs. These results demonstrate that peptide ligands conjugated to the surface of nanoparticles maintain their target specificity. This bolsters the case for peptide robustness and its persisting functionality in intracellular vehicular delivery systems.

Nanomaterial processing using self-assembly-bottom-up chemical and biological approaches.

Nanotechnology is touted as the next logical sequence in technological evolution. This has led to a substantial surge in research activities pertaining to the development and fundamental understanding of processes and assembly at the nanoscale. Both top-down and bottom-up fabrication approaches may be used to realize a range of well-defined nanostructured materials with desirable physical and chemical attributes. Among these, the bottom-up self-assembly process offers the most realistic solution toward the fabrication of next-generation functional materials and devices. Here, we present a comprehensive review on the physical basis behind self-assembly and the processes reported in recent years to direct the assembly of nanoscale functional blocks into hierarchically ordered structures. This paper emphasizes assembly in the synthetic domain as well in the biological domain, underscoring the importance of biomimetic approaches toward novel materials. In particular, two important classes of directed self-assembly, namely, (i) self-assembly among nanoparticle-polymer systems and (ii) external field-guided assembly are highlighted. The spontaneous self-assembling behavior observed in nature that leads to complex, multifunctional, hierarchical structures within biological systems is also discussed in this review. Recent research undertaken to synthesize hierarchically assembled functional materials have underscored the need as well as the benefits harvested in synergistically combining top-down fabrication methods with bottom-up self-assembly.

Nanoparticles and phage display selected peptides for imaging and therapy of cancer.

Molecular imaging probes are a special class of pharmaceuticals that target specific biochemical signatures associated with disease and allow for noninvasive imaging on the molecular level. Because changes in biochemistry occur before diseases reach an advanced stage, molecular imaging probes make it possible to locate and stage disease, track the effectiveness of drugs, treat disease, monitor response, and select patients to allow for more personalized diagnosis and treatment of disease. Targeting agents radiolabeled with positron emitters are of interest due to their ability to quantitatively measure biodistribution and receptor expression to allow for optimal dose determinations. (68)Ga is a positron emitter, which allows for quantitative imaging through positron emission chromatography (PET). The availability of (68)Ga from a generator and its ability to form stable complexes with a variety of chelates hold promise for expanding PET utilization to facilities unable to afford their own cyclotron. Nanoparticles conjugated with various proteins and peptides derived from phage display that can be selectively targeted are being developed and evaluated for guided imaging and therapy. Herein we highlight some initial efforts in combining the enhanced selectivity of nanoparticles and peptides with (68)Ga for use as molecular imaging probes.

Bombesin functionalized gold nanoparticles show in vitro and in vivo cancer receptor specificity.

Development of cancer receptor-specific gold nanoparticles will allow efficient targeting/optimum retention of engineered gold nanoparticles within tumors and thus provide synergistic advantages in oncology as it relates to molecular imaging and therapy. Bombesin (BBN) peptides have demonstrated high affinity toward gastrin-releasing peptide (GRP) receptors in vivo that are overexpressed in prostate, breast, and small-cell lung carcinoma. We have synthesized a library of GRP receptor-avid nanoplatforms by conjugating gold nanoparticles (AuNPs) with BBN peptides. Cellular interactions and binding affinities (IC(50)) of AuNP-BBN conjugates toward GRP receptors on human prostate cancer cells have been investigated in detail. In vivo studies using AuNP-BBN and its radiolabeled surrogate (198)AuNP-BBN, exhibiting high binding affinity (IC(50) in microgram ranges), provide unequivocal evidence that AuNP-BBN constructs are GRP-receptor-specific showing accumulation with high selectivity in GRP-receptor-rich pancreatic acne in normal mice and also in tumors in prostate-tumor-bearing, severe combined immunodeficient mice. The i.p. mode of delivery has been found to be efficient as AuNP-BBN conjugates showed reduced RES organ uptake with concomitant increase in uptake at tumor targets. The selective uptake of this new generation of GRP-receptor-specific AuNP-BBN peptide analogs has demonstrated realistic clinical potential in molecular imaging via x-ray computed tomography techniques as the contrast numbers in prostate tumor sites are severalfold higher as compared to the pretreatment group (Hounsfield unit = 150).

Design and development of a field applicable gold nanosensor for the detection of luteinizing hormone.

In this paper, we describe a novel strategy for the fabrication of a nanosensor for detecting luteinizing hormone (LH) of sheep using a gold nanoparticle-peptide conjugate. A new peptide sequence "CDHPPLPDILFL" (leutinizing hormone peptide, LHP) has been identified, using BLAST and Clustal W analysis, to detect antibody of LH (sheep). LHP has been synthesized and characterized, and their affinity toward anti-LH was established using enzyme linked immunosorbant assay (ELISA) technique. The thiol group in LHP directly binds with gold nanoparticles (AuNPs) to yield AuNP-LHP construct. Detailed physicochemical analysis of AuNP-LHP construct was determined using various analytical techniques. Nanosensor using gold nanoparticle peptide conjugate was developed on the basis of competitive binding of AuNP-LHP and LH toward anti-LH. Nitrocellulose membrane, precoated with anti-LH, was soaked in the mixture of AuNP-LHP and sample of analysis (LH). In the absence of LH (sheep), anti-LH coated on the membrane binds with AuNP-LHP, leading to a distinctive red color, while in the presence of LH, no color appeared in the membrane due to the interaction of anti-LH with LH thereby preventing the binding of AuNP-LHP with membrane bound anti-LH. The sensor assay developed in this study can detect LH (sheep) up to a minimal concentration of ∼50 ppm with a high degree of reproducibility and selectivity. The gold-nanoparticle-peptide based nanosensor would be a simple, portable, effective, and low cost technique for infield applications.

Bi-Functional Gold Nanorod-Protein Conjugates with Biomimetic BSA@Folic Acid Corona for Improved Tumor Targeting and Intracellular Delivery of Therapeutic Proteins in Colon Cancer 3D Spheroids.

Gold nanorods (AuNRs) remain well-developed inorganic nanocarriers of small molecules for a plethora of biomedical and therapeutic applications. However, the delivery of therapeutic proteins using AuNRs with high protein loading capacity (LC), serum stability, excellent target specificity, and minimal off-target protein release is not known. Herein, we report two bi-functional AuNR-protein nanoconjugates, AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA, supramolecularly coated with folic acid-modified BSA (BSAFA) acting as biomimetic protein corona to demonstrate targeted cytosolic delivery of enhanced green fluorescent protein (EGFP) and therapeutic ribonuclease A enzyme (RNase A) in their functional forms. AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA exhibit high LCs of ∼42 and ∼54%, respectively, increased colloidal stability, and rapid protein release in the presence of biological thiols. As a nanocarrier, AuNR@EGFP-BSAFA and AuNR@RNaseA-BSAFA show resistance to corona formation in high-serum media even after 24 h, guaranteeing a greater circulation lifetime. Folate receptor-targeting BSAFA on the AuNR surface facilitates the receptor-mediated internalization, followed by the release of EGFP and RNase A in HT29 cells. The green fluorescence dispersed throughout the cell's cytoplasm indicates successful cytosolic delivery of EGFP by AuNR@EGFP-BSAFA. AuNR@RNaseA-BSAFA-mediated therapeutic RNase A delivery in multicellular 3D spheroids of HT29 cells exhibits a radical reduction in the cellular RNA fluorescence intensity to 38%, signifying RNA degradation and subsequent cell death. The versatile nanoformulation strategy in terms of the anisotropic particle morphology, protein type, and ability for targeted delivery in the functional form makes the present AuNR-protein nanoconjugates a promising platform for potential application in cancer management.

Efficient Point-of-Care Detection of Uric Acid in the Human Blood Sample with an Enhanced Electrocatalytic Response Using Nanocomposites of Cobalt and Mixed-Valent Molybdenum Sulfide.

This work efficiently detects uric acid (UA) in a human blood sample using cobalt nanoparticle-immobilized mixed-valent molybdenum sulfide on the copper substrate in a point-of-care (PoC) device. The sensor electrode was fabricated by micromachining of Cu clad boards employing an engraver to generate a three-electrode system consisting of working electrode (WE), reference electrode (RE), and counter electrode (CE). The WE was subjected to physical vapor deposition of mixed-valent MoSx layers by a reaction between Mo(CO)6 and H2S at ∼200 °C using a simple setup following which CoNPs were electrochemically deposited. The RE and CE were covered with Ag/AgCl and Ag paste, respectively. A plasma separation membrane acted as the medium of UA/blood serum delivery to the electrodes. The material and electrochemical characterization confirmed that CoNPs over MoSx provided an enlarged electroactive surface for the direct electron transfer to achieve an enhanced electrocatalytic response. The binary combination of CoNPs and MoSx layers over the Cu electrode reduced the charge-transfer resistance by two times, enhanced the surface adsorption by more than two times, and yielded a high diffusion coefficient of 3.46 × 10-3 cm2/s. These interfacial effects facilitated the UA oxidation, leading to unprecedented mA range current density for UA sensing for the PoC device. The electrochemical detection tests in the PoC device revealed a sensitivity of 64.7 µA/µM cm-2, which is ∼50 times higher compared to the latest reported value (1.23 µA/µM cm-2), a high limit of detection of 5 nM, and shelf life of 6 months, confirming the synergistic effect-mediated high sensitivity under PoC settings. Interference tests confirmed no intervention of similar analytes. Tests on blood samples demonstrated a recovery percentage close to 100% in human serum UA, signifying the suitability of the nanocomposite-based sensor and the PoC device for clinical sensing applications.

An effective strategy for the synthesis of biocompatible gold nanoparticles using cinnamon phytochemicals for phantom CT imaging and photoacoustic detection of cancerous cells.

PURPOSE: The purpose of the present study was to explore the utilization of cinnamon-coated gold nanoparticles (Cin-AuNPs) as CT/optical contrast-enhancement agents for detection of cancer cells. METHODS: Cin-AuNPs were synthesized by a "green" procedure, and the detailed characterization was performed by physico-chemical analysis. Cytotoxicity and cellular uptake studies were carried out in normal human fibroblast and cancerous (PC-3 and MCF-7) cells, respectively. The efficacy of detecting cancerous cells was monitored using a photoacoustic technique. In vivo biodistribution was studied after IV injection of Cin-AuNPs in mice, and also a CT phantom model was generated. RESULTS: Biocompatible Cin-AuNPs were synthesized with high purity. Significant uptake of these gold nanoparticles was observed in PC-3 and MCF-7 cells. Cin-AuNPs internalized in cancerous cells facilitated detectable photoacoustic signals. In vivo biodistribution in normal mice showed steady accumulation of gold nanoparticles in lungs and rapid clearance from blood. Quantitative analysis of CT values in phantom model revealed that the cinnamon-phytochemical-coated AuNPs have reasonable attenuation efficiency. CONCLUSIONS: The results indicate that these non-toxic Cin-AuNPs can serve as excellent CT/ photoacoustic contrast-enhancement agents and may provide a novel approach toward tumor detection through nanopharmaceuticals.

New Nanomedicine Approaches Using Gold-thioguanine Nanoconjugates as Metallo-ligands.

Gold-thioguanine nanoconjugates (AuNP-TG) of size 3-4 nm were synthesized and the ratio between gold and 6-Thioguanine (TG) was estimated as ~1:1.5 using a cyanide digestion method and confirmed by flame atomic absorption spectroscopic analysis. AuNP-TG constructs showed high in vitro stability under different pH conditions and biologically relevant solutions for a period of 24 hours. Reaction of AuNP-TG with europium or platinum salts resulted in the formation of organized self-assembled metallo-networks.

Facile Detection of Blood Creatinine Using Binary Copper-Iron Oxide and rGO-Based Nanocomposite on 3D Printed Ag-Electrode under POC Settings.

Metal nanoparticles have been helpful in creatinine sensing technology under point-of-care (POC) settings because of their excellent electrocatalyst properties. However, the behavior of monometallic nanoparticles as electrochemical creatinine sensors showed limitations concerning the current density in the mA/cm2 range and wide detection window, which are essential parameters for the development of a sensor for POC applications. Herein, we report a new sensor, a reduced graphene oxide stabilized binary copper-iron oxide-based nanocomposite on a 3D printed Ag-electrode (Fe-Cu-rGO@Ag) for detecting a wide range of blood creatinine (0.01 to 1000 µM; detection limit 10 nM) in an electrochemical chip with a current density ranging between 0.185 and 1.371 mA/cm2 and sensitivity limit of 1.1 µA µM-1 cm-2 at physiological pH. Interference studies confirmed that the sensor exhibited no interference from analytes like uric acid, urea, dopamine, and glutathione. The sensor response was also evaluated to detect creatinine in human blood samples with high accuracy in less than a minute. The sensing mechanism suggested that the synergistic effects of Cu and iron oxide nanoparticles played an essential role in the efficient sensing where Fe atoms act as active sites for creatinine oxidation through the secondary amine nitrogen, and Cu nanoparticles acted as an excellent electron-transfer mediator through rGO. The rapid sensor fabrication procedure, mA/cm2 peak current density, a wide range of detection limits, low contact resistance including high selectivity, excellent linear response (R2 = 0.991), and reusability ensured the application of advanced electrochemical sensor toward the POC creatinine detection.

3-D printed electrode integrated sensing chip and a PoC device for enzyme free electrochemical detection of blood urea.

Herein we report a novel electrochemical sensing chip and a point-of-care device (PoC) for enzyme-free electrochemical detection of urea in human blood. The electrochemical sensing chip was developed by 3-D printing of conductive Ag ink and subsequent electrodeposition of AuNP-rGO nanocomposite. Material characterization of the sensing chip was conducted to find a plausible mechanism for the electrochemical reaction with urea. Subsequently, the response with varying concentrations of urea in solution and human blood samples was tested. High peak response current (~5 times than that of the highest reported value), low impedance, rapid sensor fabrication procedure, high selectivity towards urea, excellent linear response (R2 = 0.99), high sensitivity of 183 µA mM-1 cm-2, the fast response indicated by high diffusion coefficient, the limit of detection of 0.1 µM, tested shelf life of more than 6 months and recovery rate of >99% ensured the application of the developed sensor chip towards PoC urea detection test kit. A PoC device housing an electronic circuitry following the principles of linear sweep voltammetry and compatible with a sensing chip was developed. A maximum percentage error of 4.86% and maximum RSD of 3.63% confirmed the use of the PoC device for rapid urea measurements in human blood.

Advances in Thiopurine Drug Delivery: The Current State-of-the-Art.

Thiopurines (mercaptopurine, azathioprine and thioguanine) are well-established maintenance treatments for a wide range of diseases such as leukemia, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE) and other inflammatory and autoimmune diseases in general. Worldwide, millions of patients are treated with thiopurines. The use of thiopurines has been limited because of off-target effects such as myelotoxicity and hepatotoxicity. Therefore, seeking methods to enhance target-based thiopurine-based treatment is relevant, combined with pharmacogenetic testing. Controlled-release formulations for thiopurines have been clinically tested and have shown promising outcomes in inflammatory bowel disease. Latest developments in nano-formulations for thiopurines have shown encouraging pre-clinical results, but further research and development are needed. This review provides an overview of novel drug delivery strategies for thiopurines, reviewing modified release formulations and with a focus on nano-based formulations.

Nanoscale sensor design via in situ labeling of gold nanoparticles onto protein scaffolds.

A one-step method for synthesis of bioconjugated gold nanoparticles is reported. A non-toxic and biocompatible phosphorus based reducing agent was used for reduction of gold (III) and formation of nanoparticles. Physicochemical properties of protein-A stabilized gold nanoparticls were investigated. Result of immunoassay experiments confirmed the potential of the synthesized anti-protein-A conjugated gold nanoparticles for use as a simple and inexpensive test for quantitative screening of protein-A samples.

Radioactive gold nanoparticles in cancer therapy: therapeutic efficacy studies of GA-198AuNP nanoconstruct in prostate tumor-bearing mice.

Biocompatibility studies and cancer therapeutic applications of nanoparticulate beta-emitting gold-198 (198Au; beta(max) = 0.96 MeV; half-life of 2.7 days) are described. Gum arabic glycoprotein (GA)-functionalized gold nanoparticles (AuNPs) possess optimum sizes (12-18 nm core diameter and 85 nm hydrodynamic diameter) to target individual tumor cells and penetrate through tumor vasculature and pores. We report the results of detailed in vivo therapeutic investigations demonstrating the high tumor affinity of GA-198AuNPs in severely compromised immunodeficient (SCID) mice bearing human prostate tumor xenografts. Intratumoral administration of a single dose of beta-emitting GA-198AuNPs (70 Gy) resulted in clinically significant tumor regression and effective control in the growth of prostate tumors over 30 days. Three weeks after administration of GA-198AuNPs, tumor volumes for the treated animals were 82% smaller as compared with tumor volume of control group. The treatment group showed only transitory weight loss in sharp contrast to the tumor-bearing control group, which underwent substantial weight loss. Pharmacokinetic studies have provided unequivocal evidence for the optimum retention of therapeutic payload of GA-198AuNPs within the tumor site throughout the treatment regimen with minimal or no leakage of radioactivity to various nontarget organs. The measurements of white and red blood cells, platelets, and lymphocytes within the treatment group resembled those of the normal SCID mice, thus providing further evidence on the therapeutic efficacy and concomitant in vivo tolerance and nontoxic features of GA-198AuNPs. FROM THE CLINICAL EDITOR: In this study, the biocompatibility and cancer therapeutic applications of glycoprotein (GA) functionalized gold nanoparticles containing b-emitting Au-198 are described in SCID mice bearing human prostate tumor xenografts. The findings of significant therapeutic efficacy, good in vivo tolerance and non-toxic features make these particles ideal candidates for future human applications.

Gastrin releasing protein receptor specific gold nanorods: breast and prostate tumor avid nanovectors for molecular imaging.

Gastrin releasing protein receptor specific bombesin (BBN) peptide-gold nanoconjugates were successfully synthesized using gold nanorods and dithiolated peptide. The gold nanorod-bombesin (GNR-BBN) conjugates showed extraordinary in vitro stabilities against various biomolecules including NaCl, cysteine, histidine, bovine serum albumin, human serum albumin, and dithiothreitol. Quantitative measurements on the binding affinity (IC(50)) of GNR-BBN conjugates toward prostate and breast tumor cells were evaluated. The IC(50) values establish that GNR-BBN conjugates have strong affinity toward the gastrin releasing peptide receptors on both the tumors. Detailed cellular interaction studies of GNR-BBN conjugates revealed that nanorods internalize via a receptor-mediated endocytosis pathway. The receptor specific interactions of GNR-BBN conjugates provide realistic opportunities in the design and development of in vivo molecular imaging and therapy agents for cancer.

Development of 6-Thioguanine conjugated PLGA nanoparticles through thioester bond formation: Benefits of electrospray mediated drug encapsulation and sustained release in cancer therapeutic applications.

Polymeric nanoparticle-based successful delivery of hydrophobic drugs is highly desirable for its controlled and sustained release at the disease site, which is a challenge with the current synthesis methods. In the present study, an electrospray mediated facile one-step synthesis approach is explored in which a solution mixture of a hydrophobic drug, 6-thioguanine (Tg) and a biocompatible FDA approved polymer, Poly (d, l-lactide-co-glycolide) (PLGA) is injected in an applied electric field of suitable intensity to prepare drug encapsulated PLGA nanoparticles, PLGA-Tg with high yield. In order to explore the effect of external electric field on Tg loading and delivery applications, the nanoparticles are characterized using EDX, AFM, FESEM, TEM, FTIR, Raman, fluorescence, and mass spectroscopy techniques. The characterization studies indicate that the electric field mediated synthesis exhibits spherical nanoparticles with a homogenous core size distribution of ~60 nm, high encapsulation (~97.22%) and stable conjugation of Tg (via thioester linkages) with PLGA molecules in the presence of the applied electric field. The kinetic study demonstrates the 'anomalous diffusion' (non-Fickian diffusion) release mechanism in which Tg escapes from PLGA matrix with a slow, but steady diffusion rate and the sustained drug release profile continues for 60 days. To check the biological activity of the encapsulated Tg, in-vitro cell studies of the PLGA-Tg are performed on HeLa cells. The MTT assay shows significant cell death after 48 h of treatment, and the cellular internalization of the drug-loaded nanoparticles occurs through pinocytosis mediated uptake, which is established by the AFM analysis. The Raman and mass spectroscopy studies suggest that the PLGA-Tg nanoparticles are rapidly hydrolyzed inside cell cytoplasm to release Tg which initiates apoptosis-mediated cell death confirmed by as DNA fragmentation and membrane blebbing studies. The results clearly emphasize the benefits of electrospray based synthesis of polymeric nanodrug formulation through the formation of chemical bonds between polymer and drug molecules that could be easily implemented in the design and development of an effective nanotherapeutic platform with no typical 'burst effect,' prolonged release profile, and significant toxicity to the cancer cells.