Advances in biodistribution of gold nanoparticles: the influence of size, surface charge, and route of administration.

To improve the translational and clinical applications of gold nanoparticles (GNPs) in medicine there is a need for better understanding of physicochemical properties of the nanoparticles in relation to the systemic parameters andin-vivoperformance. This review presents the influence of physicochemical properties (surface charges and size) and route of administration on the biodistribution of GNPs. The role of protein corona (PC) (a unique biological identifier) as a barrier to biodistribution of GNPs, and the advances in engineered GNPs towards improving biodistribution are presented. Proteins can easily adsorb on charged (anionic and cationic) functionalized GNPs in circulation and shape the dynamics of their biodistribution. Non-ionic coatings such as PEG experience accelerated blood clearance (ABC) due to immunogenic response. While zwitterionic coatings provide stealth effects to formation of PC on the GNPs. GNPs with sizes less than 50 nm were found to circulate to several organs while the route of administration of the GNPs determines the serum protein that adsorbs on the nanoparticles.

Long-Term Accumulation, Biological Effects and Toxicity of BSA-Coated Gold Nanoparticles in the Mouse Liver, Spleen, and Kidneys.

Introduction: Gold nanoparticles are promising candidates as vehicles for drug delivery systems and could be developed into effective anticancer treatments. However, concerns about their safety need to be identified, addressed, and satisfactorily answered. Although gold nanoparticles are considered biocompatible and nontoxic, most of the toxicology evidence originates from in vitro studies, which may not reflect the responses in complex living organisms. Methods: We used an animal model to study the long-term effects of 20 nm spherical AuNPs coated with bovine serum albumin. Mice received a 1 mg/kg single intravenous dose of nanoparticles, and the biodistribution and accumulation, as well as the organ changes caused by the nanoparticles, were characterized in the liver, spleen, and kidneys during 120 days. Results: The amount of nanoparticles in the organs remained high at 120 days compared with day 1, showing a 39% reduction in the liver, a 53% increase in the spleen, and a 150% increase in the kidneys. The biological effects of chronic nanoparticle exposure were associated with early inflammatory and fibrotic responses in the organs and were more pronounced in the kidneys, despite a negligible amount of nanoparticles found in renal tissues. Conclusion: Our data suggest, that although AuNPs belong to the safest nanomaterial platforms nowadays, due to their slow tissue elimination leading to long-term accumulation in the biological systems, they may induce toxic responses in the vital organs, and so understanding of their long-term biological impact is important to consider their potential therapeutic applications.

Clinical translation of gold nanoparticles.

Gold nanoparticles display unique physicochemical features, which can be useful for therapeutic purposes. After two decades of preclinical progress, gold nanoconstructs are slowly but steadily transitioning into clinical trials. Although initially thought to be "magic golden bullets" that could be used to treat a wide range of diseases, current consensus has moved toward a more realistic approach, where gold nanoformulations are being investigated to treat specific disorders. These therapeutic applications are dictated by the pharmacokinetics and biodistribution profiles of gold nanoparticles. Here, we analyze the current clinical landscape of therapeutic gold nanoconstructs, discuss the shared characteristics that allowed for their transition from bench to bedside, and examine existing hurdles that need to be overcome before they can be approved for clinical use.

Modeling of clearance, retention, and translocation of inhaled gold nanoparticles in rats.

Objective: The increasing exposure to gold nanoparticles (AuNPs), due to their wide range of applications, has led to the need for thorough understanding of their biodistribution, following exposure. The objective of this paper is to develop a PBK model in order to study the clearance, retention and translocation of inhaled gold nanoparticles in rats, providing a basis for the understanding of the absorption, distribution, metabolism and elimination (ADME) mechanisms of AuNPs in various organs.Materials and methods: A rat PBK computational model was developed, connected to a detailed respiratory model, including the olfactory, tracheobronchial, and alveolar regions. This model was coupled with a Multiple Path Particle Dosimetry (MPPD) model to appropriately simulate the exposure to AuNPs. Three existing in vivo experimental datasets from scientific literature for the biodistribution of inhaled AuNPs for different AuNP sizes and exposure scenarios were utilized for model calibration and validation.Results and Discussion: The model was calibrated using two individual datasets for nose only inhaled and intratracheally instilled AuNPs, while an independent dataset for nose only inhaled AuNPs was used as external validation. The overall fitting over the three datasets was proved acceptable as shown by the relevant statistical metrics. The influence of several physiological parameters is also studied via a sensitivity analysis, providing useful insights into the mechanisms of NP pharmacokinetics. The key aspects of the inhaled AuNPs biodistribution are discussed, revealing the key mechanisms for the AuNPs absorption routes, the AuNP uptake by secondary organs and the influence of the AuNP size on the translocation from the lungs to blood circulation.Conclusions: The model results together with the model sensitivity analysis clarified the key mechanisms for the inhaled AuNPs biodistribution to secondary organs. It was observed that nose-only inhaled AuNPs of smaller size can enter the blood circulation through secondary routes, such as absorption through the gastrointestinal (GI) lumen, showing that such translocations should not be underestimated in biodistribution modelling. Finally, the computational framework presented in this study can be used as a basis for a more wide investigation of inhaled nanoparticles biodistribution, including interspecies extrapolation of the resulting PBK model for the inhalation and subsequent biodistribution of AuNPs in humans.

Interactions of Nanoparticles with Macrophages and Feasibility of Drug Delivery for Asthma.

Understanding the interaction between nanoparticles and immune cells is essential for the evaluation of nanotoxicity and development of nanomedicines. However, to date, there is little data on the membrane microstructure and biochemical changes in nanoparticle-loaded immune cells. In this study, we observed the microstructure of nanoparticle-loaded macrophages and changes in lipid droplets using holotomography analysis. Quantitatively analyzing the refractive index distribution of nanoparticle-loaded macrophages, we identified the interactions between nanoparticles and macrophages. The results showed that, when nanoparticles were phagocytized by macrophages, the number of lipid droplets and cell volume increased. The volume and mass of the lipid droplets slightly increased, owing to the absorption of nanoparticles. Meanwhile, the number of lipid droplets increased more conspicuously than the other factors. Furthermore, alveolar macrophages are involved in the development and progression of asthma. Studies have shown that macrophages play an essential role in the maintenance of asthma-related inflammation and tissue damage, suggesting that macrophage cells may be applied to asthma target delivery strategies. Therefore, we investigated the target delivery efficiency of gold nanoparticle-loaded macrophages at the biodistribution level, using an ovalbumin-induced asthma mouse model. Normal and severe asthma models were selected to determine the difference in the level of inflammation in the lung. Consequently, macrophages had increased mobility in models of severe asthma, compared to those of normal asthma disease. In this regard, the detection of observable differences in nanoparticle-loaded macrophages may be of primary interest, as an essential endpoint analysis for investigating nanomedical applications and immunotheragnostic strategies.

Emerging trends in the application of gold nanoformulations in colon cancer diagnosis and treatment.

Colorectal cancer is one of the most aggressive types of cancer with about two million new cases and one million deaths in 2020. The side effects of the available chemotherapies and the possibility of developing resistance against treatment highlight the importance of developing new therapeutic options. The development in the field of nanotechnology have introduced the application of nanoparticles (NPs) as a promising approach in the diagnosis and treatments of colorectal cancer and other types of cancer. Gold nanoparticles (AuNPs) are currently one of the most studied materials as they possess unique tunable properties allowing them to play a role in colorectal cancer bioimaging, diagnosis, and therapy. The high surface-to-volume ratio of AuNPs mediates their utilization in drug delivery as well as functionalization to provide specific targeting. Moreover, depending on their physical properties (size, shape), AuNPs can be modified to fit the intended application. However, there are contradictory results around the pharmacokinetics of AuNPs including their biodistribution, clearance, and toxicity. This variation of opinions is most likely due to the development of different AuNPs that vary in shape, size, and surface chemistry, in addition to the conditions under which each research was carried out. The conflicting data represent a challenge in the clinical use of AuNPs suggesting the need to understand the toxicity, fate, and long-term exposure of AuNPs in vivo. Thus, there is an unmet need for the establishment of a publicly available data base for extensive analysis. In this review, we discuss the recent advances in AuNP applications in the treatment and diagnosis of colorectal cancer, mechanisms of action, and clinical challenges.

Rapid Volumetric Optoacoustic Tracking of Nanoparticle Kinetics across Murine Organs.

Large-scale visualization of nanoparticle kinetics is essential for optimizing drug delivery and characterizing in vivo toxicity associated with engineered nanomaterials. Real-time tracking of nanoparticulate agents across multiple murine organs is hindered with the currently available whole-body preclinical imaging systems due to limitations in contrast, sensitivity, spatial, or temporal resolution. Herein, we demonstrate rapid volumetric tracking of gold nanoagent kinetics and biodistribution in mice at a suborgan level with single-sweep volumetric optoacoustic tomography (sSVOT). The imaging system accomplishes whole-body three-dimensional scans in less than 1.8 s, further attaining a high spatial resolution of 130 µm and sub-picomolar sensitivity. We visualized the clearance dynamics of purposely synthesized gold nanorods and nanorod clusters, featuring different sizes and surface chemistries as well as their corresponding accumulation within the liver and spleen. The newly discovered capacity to image rapid whole-body kinetics down to suborgan scales opens up new avenues for the development and characterization of diagnostic and therapeutic nanoagents.

Computational Studies of Au(I) and Au(III) Anticancer MetalLodrugs: A Survey.

Owing to the growing hardware capabilities and the enhancing efficacy of computational methodologies, computational chemistry approaches have constantly become more important in the development of novel anticancer metallodrugs. Besides traditional Pt-based drugs, inorganic and organometallic complexes of other transition metals are showing increasing potential in the treatment of cancer. Among them, Au(I)- and Au(III)-based compounds are promising candidates due to the strong affinity of Au(I) cations to cysteine and selenocysteine side chains of the protein residues and to Au(III) complexes being more labile and prone to the reduction to either Au(I) or Au(0) in the physiological milieu. A correct prediction of metal complexes' properties and of their bonding interactions with potential ligands requires QM computations, usually at the ab initio or DFT level. However, MM, MD, and docking approaches can also give useful information on their binding site on large biomolecular targets, such as proteins or DNA, provided a careful parametrization of the metal force field is employed. In this review, we provide an overview of the recent computational studies of Au(I) and Au(III) antitumor compounds and of their interactions with biomolecular targets, such as sulfur- and selenium-containing enzymes, like glutathione reductases, glutathione peroxidase, glutathione-S-transferase, cysteine protease, thioredoxin reductase and poly (ADP-ribose) polymerase 1.

Quantitatively Tracking Bio-Nano Interactions of Metal-Phenolic Nanocapsules by Mass Cytometry.

Polymer nanocapsules, with a hollow structure, are increasingly finding widespread use as drug delivery carriers; however, quantitatively evaluating the bio-nano interactions of nanocapsules remains challenging. Herein, poly(ethylene glycol) (PEG)-based metal-phenolic network (MPN) nanocapsules of three sizes (50, 100, and 150 nm) are engineered via supramolecular template-assisted assembly and the effect of the nanocapsule size on bio-nano interactions is investigated using in vitro cell experiments, ex vivo whole blood assays, and in vivo rat models. To track the nanocapsules by mass cytometry, a preformed gold nanoparticle (14 nm) is encapsulated into each PEG-MPN nanocapsule. The results reveal that decreasing the size of the PEG-MPN nanocapsules from 150 to 50 nm leads to reduced association (up to 70%) with phagocytic blood cells in human blood and prolongs in vivo systemic exposure in rat models. The findings provide insights into MPN-based nanocapsules and represent a platform for studying bio-nano interactions.

Assessment of ultrasmall nanocluster for early and accurate detection of atherosclerosis using positron emission tomography/computed tomography.

The development of atherosclerosis therapy is hampered by the lack of molecular imaging tools to identify the relevant biomarkers and determine the dynamic variation in vivo. Here, we show that a chemokine receptor 2 (CCR2) targeted gold nanocluster conjugated with extracellular loop 1 inverso peptide (AuNC-ECL1i) determines the initiation, progression and regression of atherosclerosis in apolipoprotein E knock-out (ApoE-/-) mouse models. The CCR2 targeted 64Cu-AuNC-ECL1i reveals sensitive detection of early atherosclerotic lesions and progression of plaques in ApoE-/- mice. CCR2 targeting specificity was confirmed by the competitive receptor blocking studies. In a mouse model of aortic arch transplantation, 64Cu-AuNC-ECL1i accurately detects the regression of plaques. Human atherosclerotic tissues show high expression of CCR2 related to the status of the disease. This study confirms CCR2 as a useful marker for atherosclerosis and points to the potential of 64Cu-AuNC-ECL1i as a targeted molecular imaging probe for future clinical translation.

Gold nanoparticle biodistribution in pregnant mice following intravenous administration varies with gestational age.

The use of nanoparticles (NPs) to deliver therapeutics to reproductive organs is an emerging approach to safely and effectively treat mothers and babies facing pregnancy complications. This study investigates the biodistribution of two different sized gold-based NPs in pregnant mice following systemic delivery as a function of gestational age. Poly(ethylene glycol)-coated 15 nm gold nanoparticles or 150 nm diameter silica core/gold nanoshells were intravenously administered to pregnant mice at gestational days (E)9.5 or 14.5. NP distribution was analyzed twenty-four hours later by inductively coupled plasma-mass spectrometry and silver staining of histological specimens. More NPs accumulated in placentas than embryos and delivery to these tissues was greater at E9.5 than E14.5. Neither NP type affected fetal weight or placental weight, indicating minimal short-term toxicity in early to mid-stage pregnancy. These findings warrant continued development of NPs as tools to deliver therapeutics to reproductive tissues safely.

Physical properties of gold nanoparticles affect skin penetration via hair follicles.

Drug penetration through the skin is significant for both transdermal and dermal delivery. One mechanism that has attracted attention over the last two decades is the transport pathway of nanoparticles via hair follicle, through the epidermis, directly to the pilosebaceous unit and blood vessels. Studies demonstrate that particle size is an important factor for drug penetration. However, in order to gain more information for the purpose of improving this mode of drug delivery, a thorough understanding of the optimal physical particle properties is needed. In this study, we fabricated fluorescently labeled gold nanoparticles (GNP) with a tight control over the size and shape. The effect of the particles' physical parameters on follicular penetration was evaluated histologically. We used horizontal human skin sections and found that the optimal size for polymeric particles is 0.25 µm. In addition, shape penetration experiments revealed gold nanostars' superiority over spherical particles. Our findings suggest the importance of the particles' physical properties in the design of nanocarriers delivered to the pilosebaceous unit.

Prostate Cancer Targeted X-Ray Fluorescence Imaging via Gold Nanoparticles Functionalized With Prostate-Specific Membrane Antigen (PSMA).

PURPOSE: The gold nanoparticle (GNP) as a promising theranostic probe has been increasingly studied. The tumor-targeting efficiency of GNPs is crucial to increase the therapeutic ratio. In this study, we developed PSMA-targeted GNPs to enhance GNP uptake in prostate cancer and developed an x-ray fluorescence imaging system to noninvasively monitor and assess GNP delivery. METHODS AND MATERIALS: For targeted therapy of prostate cancer, anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated onto PEGylated GNPs through 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) (EDC/NHS) chemistry. In vivo imaging was implemented using an in-house-developed dual-modality computed tomography (CT) and x-ray fluorescence CT (XFCT) system on mice bearing subcutaneous LNCaP prostate tumors. After intravenous administration of GNPs (15 mg/mL, 200 µL), the x-ray fluorescence signals from the tumor were collected at various time points (5 minutes to approximately 30 hours) for GNP pharmacokinetics analysis. At 24 hours after administration, x-ray fluorescence projection (XRFproj) and XFCT imaging were conducted to evaluate the prostate tumor uptake of active- and passive-targeting GNPs. Inductively coupled plasma mass spectrometry analysis was adopted as a benchmark to verify the quantification accuracy of XRFproj/XFCT imaging. RESULTS: Fluorescence microscopic imaging confirmed the enhanced (approximately 4 times) targeting efficiency of PSMA-targeted GNPs in vitro. The pharmacokinetics analysis showed enhanced tumor uptake/retention of PSMA-targeted GNPs and revealed that the peak tumor accumulation appeared at approximately 24 hours after intravenous administration. Both XRFproj and XFCT imaging presented their accuracy in quantifying GNPs within tumors noninvasively. Moreover, XFCT imaging verified its unique capabilities to simultaneously determine the heterogeneous spatial distribution and the concentration of GNPs within tumors in vivo. CONCLUSIONS: In conjunction with PSMA-targeted GNPs, XRFproj/XFCT would be a highly sensitive tool for targeted imaging of prostate cancer, benefiting the elucidation of mechanisms of GNP-assisted prostate-cancer therapy.

The cancer therapy materialization by theranostic nanoparticles based on gold doped iron oxide under electromagnetic field amplification.

The harnessing of the cancer X-ray radiation therapy by gold-decorated Fe3O4 theranostic nanoparticles (Au-Fe3O4 NPs) under electromagnetic field was articulated. The applied electromagnetic field could assemble the NPs inside cell in oriented field direction and enhance the local irradiation dose inside cell. By materializing NPs, the absorption of the energy exposed by X-ray radiation under electromagnetic field was restricted. The cytotoxic properties of the Au-Fe3O4 NPs were assessed using MTT assay in L929, HeLa and PC3 cell lines under radiation and dark conditions. The efficiency of the Au-Fe3O4 NPs under 2 Gy dose radiations was higher than 6 Gy radiations in untreated cells. The in vitro measurements showed that under electromagnetic field and X-ray radiation therapy with Au-Fe3O4 NPs, around 90% of the cancer cells population was annihilated. The in vivo measurements indicated that the tumor shape and size under X-ray with Au-Fe3O4 NPs after 3 weeks were efficiently deteriorated.

Superparamagnetic iron oxide-gold nanoparticles conjugated with porous coordination cages: Towards controlled drug release for non-invasive neuroregeneration.

This paper reports a smart intracellular nanocarrier for sustainable and controlled drug release in non-invasive neuroregeneration. The nanocarrier is composed by superparamagnetic iron oxide-gold (SPIO-Au) core-shell nanoparticles (NPs) conjugated with porous coordination cages (PCCs) through the thiol-containing molecules as bridges. The negatively charged PCC-2 and positively charged PCC-3 are compared for intracellular targeting. Both types result in intracellular targeting via direct penetration across cellular membranes. However, the pyrene (Py)-PEG-SH bridge enabled functionalization of SPIO-Au NPs with PCC-3 exhibits higher interaction with PC-12 neuron-like cells, compared with the rhodamine B (RhB)-PEG-SH bridge enabled case and the stand-alone SPIO-Au NPs. With neglectable toxicities to PC-12 cells, the proposed SPIO-Au-RhB(Py)-PCC-2(3) nanocarriers exhibit effective drug loading capacity of retinoic acid (RA) at 13.505 µg/mg of RA/NPs within 24 h. A controlled release of RA is achieved by using a low-intensity 525 nm LED light (100% compared to 40% for control group within 96 h).

[Development of Drug Delivery Technology and Nanomedicine by Using Gold Nanoparticles].

Nanomedicine is a new medical field involving the use of nanoparticles. Early examples of biocompatible nanomedicines include liposomes (Doxil®) and albumin nanoparticles (Abraxane®), and promising new nanomedicines include nanocarriers such as nanomicelles and nanoemulsions. A new trend towards the use of metal-based nanoparticles, including gold nanoparticles, has led to global clinical trials. These particles exhibit novel properties compared to conventional nanomedicines such as liposomes and albumin nanoparticles. These properties hold promise for nanomedicines, and thus the biodistribution and pharmacokinetics of metal-based nanoparticles should be carefully investigated. This had led to an increasing number of clinical trials investigating metal nanoparticles and inorganic nanoparticles. The present review evaluates multi-functional gold nanoparticles described in recent articles and shows that the unique properties of gold nanoparticles are applicable for not only drug delivery, but also for imaging. The combined therapeutic modality between therapeutics and diagnostics is called "theranostics" and is promising for future personalized cancer therapy. This review also introduces recent research from our laboratory involving the use of various kinds of molecules [polyethylene glycol (PEG), drug/cyclodextrin inclusion complexes, biosimilars and small interfering (siRNAs)] loaded onto and/or conjugated with gold nanoparticles.

Fluorine Labeling of Nanoparticles and In Vivo 19F Magnetic Resonance Imaging.

Fluorinated nanoparticles have increasing applications, but they are still challenging to prepare, especially in the case of water-soluble fluorinated nanoparticles. Herein, a fluorine labeling strategy is presented that is based on the conjugation of custom-made small fluorinated building blocks, obtained by simple synthetic transformations, with carboxylated gold nanoparticles through a convenient phase-transfer process. The synthesis of four fluorinated building blocks with different chemical shifts in 19F nuclear magnetic resonance and varied functionalities is reported, along with their conjugation onto nanoparticles. Fluorinated nanoparticles of small core size obtained by this conjugation methodology and by direct synthesis presented high transverse relaxation times (T2) ranging from 518 to 1030 ms, and a large number of equivalent fluorine atoms per nanoparticle (340-1260 fluorine atoms), which made them potential candidates for 19F magnetic resonance related applications. Finally, nontargeted fluorinated nanoparticles were probed by performing in vivo 19F magnetic resonance spectroscopy (19F MRS) in mice. Nanoparticles were detected at both 1 and 2 h after being injected. 19F MRI images were also acquired after either intravenous or subcutaneous injection. Their fate was studied by analyzing the gold content in tissues by ICP-MS. Thus, the present work provides a general fluorination strategy for nanoparticles and shows the potential use of small fluorinated nanoparticles in magnetic-resonance-related applications.

Reversing the Chirality of Surface Ligands Can Improve the Biosafety and Pharmacokinetics of Cationic Gold Nanoclusters.

Severe toxicity and rapid in vivo clearance of cationic nanomaterials seriously hinder their clinical translation. Present strategies to improve the biosafety and in vivo performance of cationic nanomaterials require neutralization of positive charge, which often compromises their efficacy. Herein, we report that substituting L-glutathione (L-GSH) on cationic gold nanoclusters (GNCs) with its D-counterpart can effectively improve the biosafety and pharmacokinetics. Compared with L-GNCs, D-GNCs do not exhibit cellular cytotoxicity, hemolysis, or acute damage to organs. Cationic D-GNCs show less cell internalization than L-GNCs, and do not induce cellular apoptosis. In vivo, the chirality of surface ligands distinctly affects the pharmacokinetics and tumor targeting abilities of D-/L-GNCs. D-GNCs show higher extended circulation time in blood plasma compared to similarly-sized and poly (ethylene glycol)-modified gold nanoparticles. This work demonstrates that the choice of chirality of surface ligands can determine toxicities and pharmacokinetics of cationic nanomaterials.

Gold Nanoparticles Perturb Drug-Metabolizing Enzymes and Antioxidants in the Livers of Male Rats: Potential Impact on Drug Interactions.

BACKGROUND AND AIM: With the wide applications of chitosan and gold nanoparticles in drug delivery and many consumer products, there is limited available information about their effects on drug-metabolizing enzymes (DMEs). Changes in DMEs could result in serious drug interactions. Therefore, this study aimed to investigate the effects of exposure to chitosan or gold nanoparticles on hepatic Phase I and II DMEs, liver function and integrity, oxidative damage and liver architecture in male rats. METHODS: Animals were divided into three equal groups: a control group, a group treated with chitosan nanoparticles (200 mg/kg, 50±5 nm) and a group treated with gold nanoparticles (4 mg/kg, 15±5 nm). Rats were orally administered their respective doses daily for 10 days. RESULTS: Both chitosan and gold nanoparticles decreased the body weights by more than 10%. Gold nanoparticles reduced the activities of antioxidants (superoxide dismutase and catalase), and reduced glutathione level and elevated the malondialdehyde level in the liver. Gold nanoparticles caused significant reductions in CYP1A1, CYP2E1, quinone oxidoreductase1, and glutathione S-transferase and elevated CYP2D6 and N-acetyl transferase2. Chitosan elevated CYP2E1 and CYP2D6 and reduced UDP-glucuronosyltransferase 1A1. Both nanoparticles disturbed the architecture of the liver, but the deleterious effects after gold nanoparticles treatment were more prominent. CONCLUSION: Taken together, gold nanoparticles severely perturbed the DMEs and would result in serious interactions with many drugs, herbs, and foods.

Redispersion of cryoaggregated gold nanoparticle by means of laser irradiation and effect on biological interactions.

Agglomeration/aggregation is an indispensable phenomenon observed by different nanoparticles. In the present study, commercial grade (50 nm) and chemically synthesized (40 nm) gold nanoparticles (AuNPs) were aggregated at sub-zero temperatures, followed by disruption of the AuNP aggregates via nanosecond laser-ablation and subsequent effect on biological interactions. AuNPs were characterized pre/post laser-ablation via UV-visible spectroscopy, transmission electron microscopy, atomic force microscopy, etc. The process of freezing (aggregation) and laser-ablation (dispersion) was performed multiple times, in order to compare the yield of nanoparticles after each cycle of laser-ablation. Further, AuNPs pre/post laser-ablation were assessed for cytotoxicity, protein-corona formation, and cell-uptake by in vitro studies using RAW264.7, Caco-2 and Neuro-2 a cell lines. Aggregates for both the types of AuNPs displayed fragmentation following first cycle of laser-ablation. In addition, AuNPs obtained after fragmentation of the aggregates showed reduction in diameter and reshaping, as compared to native AuNPs. The size and shape of the nanoparticles after second and third cycle of laser-ablation was same as that obtained after first cycle of ablation. Both laser-ablated and native AuNPs showed similar effects on viability of RAW 264.7 and Caco-2 cells, after 24 h and 48 h of exposure. Cell uptake of native and laser-ablated AuNPs was observed to be a size dependent phenomenon. Present findings showed that nanosecond laser ablation of cryoaggregated AuNPs lead to changes in the physical properties of AuNPs post ablation like size and shape, however, biological interaction with cells remained same. This work is first report on biological interactions of AuNPs generated via laser-ablation of cryoaggregated AuNPs.