Tea polyphenol-derived nanomedicine for targeted photothermal thrombolysis and inflammation suppression.

Thrombotic diseases impose a significant global health burden, and conventional drug-based thrombolytic therapies are encumbered by the risk of bleeding complications. In this study, we introduce a novel drug-free nanomedicine founded on tea polyphenols nanoparticles (TPNs), which exhibits multifaceted capabilities for localized photothermal thrombolysis. TPNs were synthesized through a one-pot process under mild conditions, deriving from the monomeric epigallocatechin-3-gallate (EGCG). Within this process, indocyanine green (ICG) was effectively encapsulated, exploiting multiple intermolecular interactions between EGCG and ICG. While both TPNs and ICG inherently possessed photothermal potential, their synergy significantly enhanced photothermal conversion and stability. Furthermore, the nanomedicine was functionalized with cRGD for targeted delivery to activated platelets within thrombus sites, eliciting robust thrombolysis upon laser irradiation across diverse thrombus types. Importantly, the nanomedicine's potent free radical scavenging abilities concurrently mitigated vascular inflammation, thus diminishing the risk of disease recurrence. In summary, this highly biocompatible multifunctional nanomaterial holds promise as a comprehensive approach that combines thrombolysis with anti-inflammatory actions, offering precision in thrombosis treatment.

Blind Spots in Development of Nanomedicines.

The field of nanomedicine demonstrates immense advantages and noteworthy expansion compared to conventional drug delivery systems like tablet, capsules, etc. Despite the innumerable advantages, it holds certain shortcomings in the form of blind spots that need to be assessed before the successful clinical translation. This perspective highlights the foremost blind spots in nanomedicine and emphasizes the challenges faced before the entry into the market, including the need for provision of safety and efficacy data by the regulatory agencies like FDA. The significant revolution of nanomedicine in the human life, particularly in patient well-being, necessitates to identify the blind spots and overcome them for effective management and treatment of ailments.

Herbal Theranostics: Controlled, Targeted Delivery and Imaging of Herbal Molecules.

Modern medicine relies on a small number of key biologics, which can be found in nature but require further characterization and purification before they can be used. Since the herbal remedy is given through a dated and ineffective method of drug administration, its effectiveness is diminished. The novel form of medicine delivery has the potential to increase the effectiveness of herbal substances while decreasing their side effects. This is the main idea behind utilising different ways of drug delivery in herbal treatments. Several benefits arise from novel formulations of herbal compounds as compared to their conventional counterparts. These include enhanced penetrating ability into tissues, constant delivery of effective doses, and resistance to physical and chemical degradation. Controlled and targeted delivery that include herbal components allow for more traditional dosing while simultaneously increasing their efficacy. Enhancing the biodistribution and target site accumulation of systemically administered herbal medicines is the goal of nanomedicine formulations. The field of nanotheranostics has made significant advancements in the development of herbal compounds by combining diagnostic and therapeutic functions on a single nanoscale platform. It is critically important to create a theranostic nanoplatform that is derived from plants and is intrinsically "all-in-one" for single molecules. In addition to examining the mechanistic approach to nanoparticle synthesis, this review highlights the therapeutic effects of nanoscale phytochemical delivery systems. Furthermore, we have evaluated the scope for future advancements in this field, discussed several nanoparticles that have been developed recently for herbal imaging, and provided experimental evidence that supports their usage.

Smart osteoclasts targeted nanomedicine based on amorphous CaCO3 for effective osteoporosis reversal.

BACKGROUND: Osteoporosis is characterized by an imbalance in bone homeostasis, resulting in the excessive dissolution of bone minerals due to the acidified microenvironment mediated by overactive osteoclasts. Oroxylin A (ORO), a natural flavonoid, has shown potential in reversing osteoporosis by inhibiting osteoclast-mediated bone resorption. The limited water solubility and lack of targeting specificity hinder the effective accumulation of Oroxylin A within the pathological environment of osteoporosis. RESULTS: Osteoclasts' microenvironment-responsive nanoparticles are prepared by incorporating Oroxylin A with amorphous calcium carbonate (ACC) and coated with glutamic acid hexapeptide-modified phospholipids, aiming at reinforcing the drug delivery efficiency as well as therapeutic effect. The obtained smart nanoparticles, coined as OAPLG, could instantly neutralize acid and release Oroxylin A in the extracellular microenvironment of osteoclasts. The combination of Oroxylin A and ACC synergistically inhibits osteoclast formation and activity, leading to a significant reversal of systemic bone loss in the ovariectomized mice model. CONCLUSION: The work highlights an intelligent nanoplatform based on ACC for spatiotemporally controlled release of lipophilic drugs, and illustrates prominent therapeutic promise against osteoporosis.

Using imaging modalities to predict nanoparticle distribution and treatment efficacy in solid tumors: The growing role of ultrasound.

Nanomedicine in oncology has not had the success in clinical impact that was anticipated in the early stages of the field's development. Ideally, nanomedicines selectively accumulate in tumor tissue and reduce systemic side effects compared to traditional chemotherapeutics. However, this has been more successful in preclinical animal models than in humans. The causes of this failure to translate may be related to the intra- and inter-patient heterogeneity of the tumor microenvironment. Predicting whether a patient will respond positively to treatment prior to its initiation, through evaluation of characteristics like nanoparticle extravasation and retention potential in the tumor, may be a way to improve nanomedicine success rate. While there are many potential strategies to accomplish this, prediction and patient stratification via noninvasive medical imaging may be the most efficient and specific strategy. There have been some preclinical and clinical advances in this area using MRI, CT, PET, and other modalities. An alternative approach that has not been studied as extensively is biomedical ultrasound, including techniques such as multiparametric contrast-enhanced ultrasound (mpCEUS), doppler, elastography, and super-resolution processing. Ultrasound is safe, inexpensive, noninvasive, and capable of imaging the entire tumor with high temporal and spatial resolution. In this work, we summarize the in vivo imaging tools that have been used to predict nanoparticle distribution and treatment efficacy in oncology. We emphasize ultrasound imaging and the recent developments in the field concerning CEUS. The successful implementation of an imaging strategy for prediction of nanoparticle accumulation in tumors could lead to increased clinical translation of nanomedicines, and subsequently, improved patient outcomes. This article is categorized under: Diagnostic Tools In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery Nanomedicine for Oncologic Disease Therapeutic Approaches and Drug Discovery Emerging Technologies.

How Precise are Nanomedicines in Overcoming the Blood-Brain Barrier? A Comprehensive Review of the Literature.

New nanotechnology strategies for enhancing drug delivery in brain disorders have recently received increasing attention from drug designers. The treatment of neurological conditions, including brain tumors, stroke, Parkinson's Disease (PD), and Alzheimer's disease (AD), may be greatly influenced by nanotechnology. Numerous studies on neurodegeneration have demonstrated the effective application of nanomaterials in the treatment of brain illnesses. Nanocarriers (NCs) have made it easier to deliver drugs precisely to where they are needed. Thus, the most effective use of nanomaterials is in the treatment of various brain diseases, as this amplifies the overall impact of medication and emphasizes the significance of nanotherapeutics through gene therapy, enzyme replacement therapy, and blood-barrier mechanisms. Recent advances in nanotechnology have led to the development of multifunctional nanotherapeutic agents, a promising treatment for brain disorders. This novel method reduces the side effects and improves treatment outcomes. This review critically assesses efficient nano-based systems in light of obstacles and outstanding achievements. Nanocarriers that transfer medications across the blood-brain barrier and nano-assisted therapies, including nano-immunotherapy, nano-gene therapy, nano enzyme replacement therapy, scaffolds, and 3D to 6D printing, have been widely explored for the treatment of brain disorders. This study aimed to evaluate existing literature regarding the use of nanotechnology in the development of drug delivery systems that can penetrate the blood-brain barrier (BBB) and deliver therapeutic agents to treat various brain disorders.

Magnetic Hydroxyapatite Nanoparticles in Regenerative Medicine and Nanomedicine.

This review focuses on the latest advancements in magnetic hydroxyapatite (mHA) nanoparticles and their potential applications in nanomedicine and regenerative medicine. mHA nanoparticles have gained significant interest over the last few years for their great potential, offering advanced multi-therapeutic strategies because of their biocompatibility, bioactivity, and unique physicochemical features, enabling on-demand activation and control. The most relevant synthetic methods to obtain magnetic apatite-based materials, either in the form of iron-doped HA nanoparticles showing intrinsic magnetic properties or composite/hybrid compounds between HA and superparamagnetic metal oxide nanoparticles, are described as highlighting structure-property correlations. Following this, this review discusses the application of various magnetic hydroxyapatite nanomaterials in bone regeneration and nanomedicine. Finally, novel perspectives are investigated with respect to the ability of mHA nanoparticles to improve nanocarriers with homogeneous structures to promote multifunctional biological applications, such as cell stimulation and instruction, antimicrobial activity, and drug release with on-demand triggering.

Recent advances in multifunctional dendrimer-based complexes for cancer treatment.

The application of nanotechnology in biological and medical fields have resulted in the creation of new devices, supramolecular systems, structures, complexes, and composites. Dendrimers are relatively new nanotechnological polymers with unique features; they are globular in shape, with a topological structure formed by monomeric subunit branches diverging to the sides from the central nucleus. This review analyzes the main features of dendrimers and their applications in biology and medicine regarding cancer treatment. Dendrimers have applications that include drug and gene carriers, antioxidant agents, imaging agents, and adjuvants, but importantly, dendrimers can create complex nanosized constructions that combine features such as drug/gene carriers and imaging agents. Dendrimer-based nanosystems include different metals that enhance oxidative stress, polyethylene glycol to provide biosafety, an imaging agent (a fluorescent, radioactive, magnetic resonance imaging probe), a drug or/and nucleic acid that provides a single or dual action on cells or tissues. One of major benefit of dendrimers is their easy release from the body (in contrast to metal nanoparticles, fullerenes, and carbon nanotubes), allowing the creation of biosafe constructions. Some dendrimers are already clinically approved and are being used as drugs, but many nanocomplexes are currently being studied for clinical practice. In summary, dendrimers are very useful tool in the creation of complex nanoconstructions for personalized nanomedicine. This article is categorized under: Diagnostic Tools > Diagnostic Nanodevices Diagnostic Tools > In Vivo Nanodiagnostics and Imaging Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.

Nanomedicine and nanobiotechnology in India.

Nanomedicine, an interdisciplinary field combining nanotechnology and medicine, has gained immense attention in recent years due to its potential in revolutionizing healthcare. India, being an emerging hub for scientific research and development, has made significant strides in nanomedicine research. This special issue is dedicated to the exciting research that are being conducted by the leading Indian scientists in various Indian institutions. This article is categorized under: Biology-Inspired Nanomaterials > Lipid-Based Structures Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Coassembly Nanomedicine Mediated by Intermolecular Interactions Between Methotrexate and Baricitinib for Improved Rheumatoid Arthritis Treatment.

The combination of anti-rheumatoid arthritis (RA) drugs methotrexate (MTX) and baricitinib (BTN) has been reported to improve RA treatment efficacy. However, study on the strategy of combination is elusive when considering the benefit of the synergy between MTX and BTN. In this study, we found that the N-heterocyclic rings in the MTX and BTN offer hydrogen bonds and π-π stacking interactions, driving the formation of exquisite vesicular morphology of nanovesicles, denoted as MB NVs. The MB NVs with the MTX/BTN weight ratio of 2:1, MB NVs (2:1), showed an improved anti-RA effect through the synergy between the anti-inflammatory and antiproliferative responses. This work presents that the intermolecular interactions between drug molecules could mediate the coassembly behavior into nanomedicine as well as the therapy synergy both in vitro and in vivo, which may provide further understanding on the rational design of combination nanomedicine for therapeutic purposes.

Programming Intracellular Clustering of Spiky Nanoparticles via Liposome Encapsulation.

The intracellular clustering of anisotropic nanoparticles is crucial to the improvement of the localized surface plasmon resonance (LSPR) for phototherapy applications. Herein, we programmed the intracellular clustering process of spiky nanoparticles (SNPs) by encapsulating them into an anionic liposome via a frame-guided self-assembly approach. The liposome-encapsulated SNPs (lipo-SNPs) exhibited distinct and enhanced lysosome-triggered aggregation behavior while maintaining excellent monodispersity, even in acidic or protein-rich environments. We explored the enhancement of the photothermal therapy performance for SNPs as a proof of concept. The photothermal conversion efficiency of lipo-SNPs clusters significantly increased 15 times compared to that of single lipo-SNPs. Upon accumulation in lysosomes with a 2.4-fold increase in clustering, lipo-SNPs resulted in an increase in cell-killing efficiency to 45% from 12% at 24 µg/mL. These findings indicated that liposome encapsulation provides a promising approach to programing nanoparticle clustering at the target site, which facilitates advances in the development of smart nanomedicine with programmable enhancement in LSPR.

Antiangiogenic Therapeutic mRNA Delivery Using Lung-Selective Polymeric Nanomedicine for Lung Cancer Treatment.

Therapeutic antibodies that block vascular endothelial growth factor (VEGF) show clinical benefits in treating nonsmall cell lung cancers (NSCLCs) by inhibiting tumor angiogenesis. Nonetheless, the therapeutic effects of systemically administered anti-VEGF antibodies are often hindered in NSCLCs because of their limited distribution in the lungs and their adverse effects on normal tissues. These challenges can be overcome by delivering therapeutic antibodies in their mRNA form to lung endothelial cells, a primary target of VEGF-mediated pulmonary angiogenesis, to suppress the NSCLCs. In this study, we synthesized derivatives of poly(ß-amino esters) (PBAEs) and prepared nanoparticles to encapsulate the synthetic mRNA encoding bevacizumab, an anti-VEGF antibody used in the clinic. Optimization of nanoparticle formulations resulted in a selective lung transfection after intravenous administration. Notably, the optimized PBAE nanoparticles were distributed in lung endothelial cells, resulting in the secretion of bevacizumab. We analyzed the protein corona on the lung- and spleen-targeting nanoparticles using proteomics and found distinctive features potentially contributing to their organ-selectivity. Lastly, bevacizumab mRNA delivered by the lung-targeting PBAE nanoparticles more significantly inhibited tumor proliferation and angiogenesis than recombinant bevacizumab protein in orthotopic NSCLC mouse models, supporting the therapeutic potential of bevacizumab mRNA therapy and its selective delivery through lung-targeting nanoparticles. Our proof-of-principle results highlight the clinical benefits of nanoparticle-mediated mRNA therapy in anticancer antibody treatment in preclinical models.

Nanomedicines Promote Cartilage Regeneration in Osteoarthritis by Synergistically Enhancing Chondrogenesis of Mesenchymal Stem Cells and Regulating Inflammatory Environment.

Osteoarthritis (OA) is a degenerative joint disease characterized by progressive erosion of the articular cartilage and inflammation. Mesenchymal stem cells' (MSCs) transplantation in OA treatment is emerging, but its clinical application is still limited by the low efficiency in oriented differentiation. In our study, to improve the therapeutic efficiencies of MSCs in OA treatment by carbonic anhydrase IX (CA9) siRNA (siCA9)-based inflammation regulation and Kartogenin (KGN)-based chondrogenic differentiation, the combination strategy of MSCs and the nanomedicine codelivering KGN and siCA9 (AHK-CaP/siCA9 NPs) was used. In vitro results demonstrated that these NPs could improve the inflammatory microenvironment through repolarization of M1 macrophages to the M2 phenotype by downregulating the expression levels of CA9 mRNA. Meanwhile, these NPs could also enhance the chondrogenesis of bone marrow-derived mesenchymal stem cells (BMSCs) by upregulating the pro-chondrogenic TGF-ß1, ACAN, and Col2α1 mRNA levels. Moreover, in an advanced OA mouse model, compared with BMSCs alone group, the lower synovitis score and OARSI score were found in the group of BMSCs plus AHK-CaP/siCA9 NPs, suggesting that this combination approach could effectively inhibit synovitis and promote cartilage regeneration in OA progression. Therefore, the synchronization of regulating the inflammatory microenvironment through macrophage reprogramming (CA9 gene silencing) and promoting MSCs oriented differentiation through a chondrogenic agent (KGN) may be a potential strategy to maximize the therapeutic efficiency of MSCs for OA treatment.

New insights into nanomedicines for oral delivery of glucagon-like peptide-1 analogs.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder that arises when the body cannot respond fully to insulin, leading to impaired glucose tolerance. Currently, the treatment embraces non-pharmacological actions (e.g., diet and exercise) co-associated with the administration of antidiabetic drugs. Metformin is the first-line treatment for T2DM; nevertheless, alternative therapeutic strategies involving glucagon-like peptide-1 (GLP-1) analogs have been explored for managing the disease. GLP-1 analogs trigger insulin secretion and suppress glucagon release in a glucose-dependent manner thereby, reducing the risk of hyperglycemia. Additionally, GLP-1 analogs have an extended plasma half-life compared to the endogenous peptide due to their high resistance to degradation by dipeptidyl peptidase-4. However, GLP-1 analogs are mainly administered via subcutaneous route, which can be inconvenient for the patients. Even considering an oral delivery approach, GLP-1 analogs are exposed to the harsh conditions of the gastrointestinal tract (GIT) and the intestinal barriers (mucus and epithelium). Hereupon, there is an unmet need to develop non-invasive oral transmucosal drug delivery strategies, such as the incorporation of GLP-1 analogs into nanoplatforms, to overcome the GIT barriers. Nanotechnology has the potential to shield antidiabetic peptides against the acidic pH and enzymatic activity of the stomach. In addition, the nanoparticles can be coated and/or surface-conjugated with mucodiffusive polymers and target intestinal ligands to improve their transport through the intestinal mucus and epithelium. This review focuses on the main hurdles associated with the oral administration of GLP-1 and GLP-1 analogs, and the nanosystems developed to improve the oral bioavailability of the antidiabetic peptides. This article is categorized under: Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease Nanotechnology Approaches to Biology > Nanoscale Systems in Biology.

Long-Acting Heterodimeric Paclitaxel-Idebenone Prodrug-Based Nanomedicine Promotes Functional Recovery after Spinal Cord Injury.

After spinal cord injury (SCI), successive systemic administration of microtubule-stabilizing agents has been shown to promote axon regeneration. However, this approach is limited by poor drug bioavailability, especially given the rapid restoration of the blood-spinal cord barrier. There is a pressing need for long-acting formulations of microtubule-stabilizing agents in treating SCI. Here, we conjugated the antioxidant idebenone with microtubule-stabilizing paclitaxel to create a heterodimeric paclitaxel-idebenone prodrug via an acid-activatable, self-immolative ketal linker and then fabricated it into chondroitin sulfate proteoglycan-binding nanomedicine, enabling drug retention within the spinal cord for at least 2 weeks and notable enhancement in hindlimb motor function and axon regeneration after a single intraspinal administration. Additional investigations uncovered that idebenone can suppress the activation of microglia and neuronal ferroptosis, thereby amplifying the therapeutic effect of paclitaxel. This prodrug-based nanomedicine simultaneously accomplishes neuroprotection and axon regeneration, offering a promising therapeutic strategy for SCI.

Recent advances in nanomaterial-driven strategies for diagnosis and therapy of vascular anomalies.

Nanotechnology has demonstrated immense potential in various fields, especially in biomedical field. Among these domains, the development of nanotechnology for diagnosing and treating vascular anomalies has garnered significant attention. Vascular anomalies refer to structural and functional anomalies within the vascular system, which can result in conditions such as vascular malformations and tumors. These anomalies can significantly impact the quality of life of patients and pose significant health concerns. Nanoscale contrast agents have been developed for targeted imaging of blood vessels, enabling more precise identification and characterization of vascular anomalies. These contrast agents can be designed to bind specifically to abnormal blood vessels, providing healthcare professionals with a clearer view of the affected areas. More importantly, nanotechnology also offers promising solutions for targeted therapeutic interventions. Nanoparticles can be engineered to deliver drugs directly to the site of vascular anomalies, maximizing therapeutic effects while minimizing side effects on healthy tissues. Meanwhile, by incorporating functional components into nanoparticles, such as photosensitizers, nanotechnology enables innovative treatment modalities such as photothermal therapy and photodynamic therapy. This review focuses on the applications and potential of nanotechnology in the imaging and therapy of vascular anomalies, as well as discusses the present challenges and future directions.

Nanomedicine alleviates doxorubicin-induced cardiotoxicity and enhances chemotherapy synergistic chemodynamic therapy.

Doxorubicin (DOX) is widely used in clinic as a broad-spectrum chemotherapy drug, which can enhance the efficacy of chemodynamic therapy (CDT) by interfering tumor-related metabolize to increase H2O2 content. However, DOX can induce serious cardiomyopathy (DIC) due to its oxidative stress in cardiomyocytes. Eliminating oxidative stress would create a significant opportunity for the clinical application of DOX combined with CDT. To address this issue, we introduced sodium ascorbate (AscNa), the main reason is that AscNa can be catalyzed to produce H2O2 by the abundant Fe3+ in the tumor site, thereby enhancing CDT. While the content of Fe3+ in heart tissue is relatively low, so the oxidation of AscNa had tumor specificity. Meanwhile, due to its inherent reducing properties, AscNa could also eliminate the oxidative stress generated by DOX, preventing cardiotoxicity. Due to the differences between myocardial tissue and tumor microenvironment, a novel nanomedicine was designed. MoS2 was employed as a carrier and CDT catalyst, loaded with DOX and AscNa, coating with homologous tumor cell membrane to construct an acid-responsive nanomedicine MoS2-DOX/AscNa@M (MDA@M). In tumor cells, AscNa enhances the synergistic therapy of DOX and MoS2. In cardiomyocytes, AscNa could effectively reduce the cardiomyopathy induced by DOX. Overall, this study enhanced the clinical potential of chemotherapy synergistic CDT.

Combination drug therapy by herbal nanomedicine prevent multidrug resistance protein 1: promote apoptosis in Lung Carcinoma.

Given the numerous adverse effects of lung cancer treatment, more research on non-toxic medications is urgently needed. Curcumin (CUR) and berberine (BBR) combat drug resistance by controlling the expression of multidrug resistant pump (MDR1). Fascinatingly, combining these medications increases the effectiveness of preventing lung cancer. Their low solubility and poor stability, however, restrict their therapeutic efficacy. Because of the improved bioavailability and increased encapsulation effectiveness of water-insoluble medicines, surfactant-based nanovesicles have recently received a great deal of attention. The current study sought to elucidate the Combination drug therapy by herbal nanomedicine prevent multidrug resistance protein 1: promote apoptosis in Lung Carcinoma. The impact of several tween (20, 60, and 80) types with varied hydrophobic tails on BBR/CUR-TNV was evaluated. Additionally, the MDR1 activity and apoptosis rate of the BBR/CUR-TNV combination therapy were assessed. The encapsulation effectiveness of TNV was affected by the type of tween. With the TNV made from tween 60, cholesterol, and PEG (47.5: 47.5:5), more encapsulation effectiveness was attained. By combining CUR with BBR, especially when given in TNV, apoptosis increased. Additionally, when CUR and BBR were administered in combination, they significantly reduced the risk of MDR1 development. The current work suggests that the delivery of berberine and curcumin as a combination medication therapy via tween-based nanovesicles may be a potential lung cancer treatment.

Advancements in Nanoparticle Characterization.

Nanotechnology for drug delivery has made significant advancements over the last two decades. Innovations have been made in cancer research and development, including chemotherapies, imaging agents, and vaccine strategies, as well as other therapeutic areas, e.g., the recent commercialization of mRNA lipid nanoparticles as vaccines against the SARS-CoV-2 virus. The field has also seen technological advancements to aid in addressing the complex questions posed by these novel therapies. In this latest edition of protocols and methods for nanoparticle characterization, we highlight both old and new methodologies for defining physicochemical properties, present both in vitro and in vivo methods to test for a variety of immunotoxicities, and describe assays used for pharmacological studies to assess drug release and tissue distribution.

Evaluation of Nanomedicine Tissue Distribution and Stability by Alexa Fluor 488 and PEG Immunohistochemistry.

A primary issue with nanomedicine biological evaluation is determination of nanoparticle carrier tissue distribution and stability. Here we present a method to evaluate nanomedicine distribution in tissues that is applicable to most nanomedicine constructs. This method utilizes immunohistochemical (IHC) analysis of an Alexa Fluor 488-tag and/or polyethylene glycol (PEG), a very common nanomedicine component, for tissue localization. Using specific Alexa Fluor 488- and/or PEG antibody-based IHC staining procedures allows evaluation of high-resolution nanoparticle tissue distribution, nanoparticle tissue stability, and also allows correlation of distribution with morphological changes. This protocol outlines the methods to follow to ensure proper tissue collection and optimized immunohistochemical staining of Alexa Fluor 488-tag and PEG in tissues.