Delivery by Cationic Gelatin Nanoparticles Strongly Increases the Immunostimulatory Effects of CpG Oligonucleotides

Purpose Cationized gelatin nanoparticles (GNPs) were used as carrier to improve delivery of immunostimulatory CpG oligonucleotides (CpG ODN) both in vitro and in vivo. Methods Uptake of CpG ODN-loaded cationized gelatin nanoparticles (CpG-GNPs) into murine myeloid dendritic cells (DCs) and their respective immunostimulatory activity was monitored. In vivo, induction of cytokine secretion by CpG-GNPs was measured. For experiments on primary human cells, prototypes of the three CpG ODN classes were adsorbed onto GNPs. Uptake and induction of proinflammatory cytokines were assessed in human plasmacytoid DCs and B cells, the only existing human target cells for CpG ODN. Results In the murine system, gelatin nanoparticle formulations enhanced the uptake and immunostimulatory activity of CpG ODN both in vitro and in vivo. Furthermore, delivery by cationized gelatin nanoparticles of CpG ODN of the classes B and C to primary human plasmacytoid DCs increased production of IFN-α, a key cytokine in the driving of both the innate and adaptive immune responses. Conclusion GNPs can be used as a biodegradable and well tolerated carrier to deliver CpG ODN to their target cells and strongly increase activation of the immune system. This concept may be applied as novel adjuvant for antiviral and antitumoral vaccines.     

Nanoparticle-based immunopotentiation via tetanus toxoid-loaded gelatin and aminated gelatin nanoparticles

Gelatin nanoparticles (GNPs) and aminated gelatin nanoparticles (AGNPs) were prepared and used as an adjuvant to improve the delivery of tetanus toxoid (TT). Nanoparticles were characterized in vitro for their size, shape, entrapment, and release. TT-FITC conjugate was used to determine entrapment and release from nanoparticles. The immune-stimulating activity was studied by measuring anti-TT IgG, IgG1, and IgG2a isotype and cytokine responses following subcutaneous (s.c) injection of nanoparticles in BALB/c mice and was compared with alum-TT vaccine. Gelatin and aminated gelatin (AG) specific IgG response was also determined. Both GNPs and AGNPs demonstrated comparable IgG response and a significantly higher (p?< 0.05) cytokine response (IL-2 and IFN γ) as compared to alum-TT vaccine. Nanoparticulate formulations elicited both Th1 and Th2 responses and induced negligible undesirable immunogenicity against the carrier, as demonstrated by lower level of gelatin and AG-specific IgG response as compared to control.     

Mannosylated gelatin nanoparticles bearing isoniazid for effective management of tuberculosis

The mannosylated gelatin nanoparticles (Mn-GNPs) were prepared for the selective delivery of an antitubercular drug, isoniazid (INH), to the alveolar macrophages. The gelatin nanoparticles (GNPs) were prepared by using a two-step desolvation method and efficiently conjugated with mannose. Various parameters such as particle size, polydispersity index, zeta potential, % entrapment efficiency, in vitro drug release, macrophage uptake, in vivo biodistribution, antitubercular activity and hepatotoxicity of plain and Mn-GNPs were determined. The size of nanoparticles (both plain and Mn-GNPs) was found to be in range of 260–380?nm, and maximum drug payload was found to be 40–55%. Average particle size of Mn-GNPs was more, whereas drug entrapment was lesser compared to plain GNPs. The organ distribution studies demonstrated the efficiency of Mn-GNPs for spatial delivery of INH to alveolar tissues. Intravenous administration of INH loaded Mn-GNPs (I-Mn-GNPs) resulted in significant reduction in bacterial counts in the lungs and spleen of tuberculosis-infected (TB-infected) mice and also reduction in the hepatotoxicity of the drug. This study revealed that mannose conjugated GNPs may be explored as potential carrier for safer and efficient management of TB through targeted delivery of INH when compared to plain GNPs and free drug.     

Nanoparticle-based immunopotentiation via tetanus toxoid-loaded gelatin and aminated gelatin nanoparticles

Gelatin nanoparticles (GNPs) and aminated gelatin nanoparticles (AGNPs) were prepared and used as an adjuvant to improve the delivery of tetanus toxoid (TT). Nanoparticles were characterized in vitro for their size, shape, entrapment, and release. TT-FITC conjugate was used to determine entrapment and release from nanoparticles. The immune-stimulating activity was studied by measuring anti-TT IgG, IgG1, and IgG2a isotype and cytokine responses following subcutaneous (s.c) injection of nanoparticles in BALB/c mice and was compared with alum-TT vaccine. Gelatin and aminated gelatin (AG) specific IgG response was also determined. Both GNPs and AGNPs demonstrated comparable IgG response and a significantly higher (p?<?0.05) cytokine response (IL-2 and IFN γ) as compared to alum-TT vaccine. Nanoparticulate formulations elicited both Th1 and Th2 responses and induced negligible undesirable immunogenicity against the carrier, as demonstrated by lower level of gelatin and AG-specific IgG response as compared to control.     

Towards an inhalative in vivo application of immunomodulating gelatin nanoparticles in horse-related preformulation studies

Delivering active ingredients using biocompatible and biodegradable carriers such as gelatin nanoparticles (GNPs) to the lung constitutes a promising non-invasive route of administration. However, the pulmonary delivery of nanoparticle-based immunotherapy is still a field that requires more clarification.

In this study, GNPs loaded with cytosine-phosphate-guanine oligodeoxynucleotides (CpG-ODN)-loaded and plain GNPs were aerosolised either by a conventional pressured metered dose inhaler (pMDI) or by active or passive vibrating-mesh (VM) nebulisers. GNP sizes after nebulisation by active and passive VM nebulisers were 248.2?±?7.34 and 222.3?±?1.42?nm, respectively. GNP concentrations after aerosolisation were found consistent and second-stage particle deposition in an impinger was up to 65.68?±?11.2% of the nebulised dose. VM nebulisers produced high fine particle fractions, while pMDIs did not. Nebulised CpG-ODN-loaded GNPs remained capable to stimulate IL-10 release (225.2?±?56.3?pg/ml) in vitro from equine alveolar lymphocytes. Thus, a novel system for pulmonary GNP-mediated immunotherapy in vivo was established.     

Development of surface-functionalised nanoparticles for FGF2 receptor-based solid tumour targeting

The surface-functionalised gelatin nanoparticles (GNPs) containing cisplatin were developed and characterised for breast cancer targeting using fibroblast growth factor-2 (FGF2) receptors which are overexpressed on breast cancer cells. The GNPs were prepared using two-step desolvation method and then the surface of GNPs was functionalised with activated heparin. They were characterised for surface morphology, particle size and size distribution, surface charge, entrapment efficiency and in?vitro drug release. The results revealed that the mean diameter of GNPs was 173?±?2.2?nm with smooth surface, which was increased to 189?±?3.4?nm after coupling with heparin (H-GNPs). The targeting effect of H-GNPs and GNPs was investigated by in?vitro cell uptake study on human breast cancer MDA-MB-231 cell line, which exhibited greater uptake of H-GNPs as compared to GNPs. Therefore, it is suggested that H-GNPs can be used as an effective carrier for solid tumour targeting.     

Exploring gold nanoparticles interaction with mucins: A spectroscopic-based study

The interaction between two mucin types (mucin from porcine stomach – PGM and mucin from bovine submaxillary glands – BSM) and gold nanoparticles (GNPs) of various size (5, 20 and 40?nm) and functionalization (with cysteamine or thioglycolic acid) was studied under physiological conditions, in order to investigate the affinity of the nanoparticles to the proteins.Different methods are employed to monitor the interactions: UV–vis and fluorescence spectroscopy, fluorescence lifetime, circular dichroism and transmission electron microscopy. These studies have shown the formation of a complex between GNPs and both PGM and BSM.This aspect could be of great importance for the use of gold nanoparticles for biomedical purposes in those diseases where qualitative and quantitative mucin anomalies play an essential role in mucus composition and rheology.     

Antitumor activity of intratracheal inhalation of temozolomide (TMZ) loaded into gold nanoparticles and/or liposomes against urethane-induced lung cancer in BALB/c mice

The current study aimed to develop gold nanoparticles (GNPs) and liposome-embedded gold nanoparticles (LGNPs) as drug carriers for temozolomide (TMZ) and investigate the possible therapeutic effects of intratracheal inhalation of nanoformulation of TMZ-loaded gold nanoparticles (TGNPs) and liposome-embedded TGNPs (LTGNPs) against urethane-induced lung cancer in BALB/c mice. Physicochemical characters and zeta potential studies for gold nanoparticles (GNPs) and liposome-embedded gold nanoparticles (LGNPs) were performed. The current study was conducted by inducing lung cancer chemically via repeated exposure to urethane in BALB/C mice. GNPs and LGNPs were exhibited in uniform spherical shape with adequate dispersion stability. GNPs and LGNPs showed no significant changes in comparison to control group with high safety profile, while TGNPs and LTGNPs succeed to improve all biochemical data and histological patterns. GNPs and LGNPs are promising drug carriers and succeeded in the delivery of small and efficient dose of temozolomide in treatment lung cancer. Antitumor activity was pronounced in animal-treated LTGNPs, these effects may be due to synergistic effects resulted from combination of temozolomide and gold nanoparticles and liposomes that may improve the drug distribution and penetration.     

Exploiting 4-sulphate N-acetyl galactosamine decorated gelatin nanoparticles for effective targeting to professional phagocytes in vitro and in vivo

The present study was focused on the development of surface modified gelatin nanoparticles (SGNPs) using novel ligand 4-sulfated N-acetyl galactosamine (4-SO₄GalNAc) for specific targeting to macrophages. The gelatin has been modified with the potential targeting moiety 4-SO₄GalNAc, which was further used for the preparation of modified nanoparticles. The nanoparticles have been prepared by two step desolvation method. The SGNPs and unmodified gelatin nanoparticles (GNPs) were loaded with doxorubicin (DxR) and its targeting potential was compared. Developed DxR-loaded SGNPs (DxR-SGNPs) were found to have negative zeta potential (?19.8?±?0.22 mV) whereas DxR-loaded GNPs (DxR-GNPs) have the positive zeta potential of around +12.2?±?0.36 mV. The mean particle size of DxR-SGNPs and DxR-GNPs was found to be 283?±?7 and 134?±?5?nm, respectively. Flow cytometric data confirmed the enhanced uptake of DxR-SGNPs in J774A.1 and PBMC when compared with DxR-GNPs. Intracellular localization studies indicate that the fluorescence intensity of DxR-SGNPs was significantly higher when compared to DxR-GNPs. DxR-SGNPs rendered significantly higher localization of DxR in liver and spleen as compared to DxR-GNPs after i.v. administration. The study stipulates that 4-SO₄GalNAc assures for targeting resident macrophages.     

Cellular uptake and nanoscale localization of gold nanoparticles in cancer using label-free confocal Raman microscopy

This work demonstrates the use of confocal Raman microscopy (CRM) to measure the dynamics of cellular uptake and localization of gold nanoparticles (GNP) with nanoscale resolution. This is important as nanoparticle cellular interactions are increasingly under investigation to support applications as diverse as drug delivery, gene transfection and a variety of heat and radiation based therapeutics. At the heart of these applications is a need to know the dynamics of nanoparticle cellular uptake and localization (i.e., cell membrane, cytoplasm or nucleus). This process can change dramatically based on size, charge, shape and ligand attached to the nanoparticle. While electron microscopy, atomic emission spectroscopy and histology can be used to assess cellular uptake, they are labor intensive and post-mortem and can miss critical dynamics of the process. For this reason investigators are increasingly turning to optically active nanoparticles that allow direct microscopic interrogation of uptake. Here we show that CRM adds to this evolving armamentarium as a fast, noninvasive, and label-free technique to dynamically study cellular uptake of GNPs with subcellular detail in cancer. Raman laser interaction with GNPs inside cells shows unique spectroscopic features corresponding to the intracellular localization of GNPs over 2 to 24 h at the membrane, cytoplasm or nucleus that are separately verified by histology (silver staining) and electron microscopy. These results show that CRM has the potential to facilitate high-throughput study of the dynamics and localization of a variety of GNPs in multiple cell types.     

Protein seeding of gold nanoparticles and mechanism of glycation sensing

The plasmon resonance of gold nanoparticles (GNPs) synthesized on a protein template senses formation of advanced glycosylated end products (AGEs). A graded alteration of plasmon resonance (both the peak and intensity are affected) is observed as the glycation progresses. Transmission electron microscopy shows significant shift of the size distribution of GNPs in presence of glycation. The higher plasmon resonance is thus caused by increased formation of GNPs, which in turn is attributed to a larger number of smaller particles. To study the binding of the protein with the GNP, infrared (IR) spectroscopy and circular dichroism (CD) studies were undertaken. Whereas the CD studies confirmed the emergence of beta-structure and loss of alpha-helix, the IR data indicated glycation-induced alterations in the amide I region. The proposed sensor for formation of AGEs thus apparently operates by direct or indirect conjugation with amino groups. Incidentally, glycation and AGE formation are responsible for a number of diabetes-related clinical conditions, and the present approach could be adopted for use for a simple colorimetric assay for the AGEs.     

Mannosylated gelatin nanoparticles bearing an anti-HIV drug didanosine for site-specific delivery

The present investigation was aimed at developing and exploring the use of mannosylated gelatin nanoparticles for the selective delivery of an anti-HIV drug, didanosine, to the target organs. The mannosylated gelatin nanoparticles (MN-G-NPs) were prepared using a two-step desolvation technique and coupled with mannose using the amino group of gelatin present on the surface of nanoparticles. The mannosylation was confirmed using infrared and nuclear magnetic resonance spectroscopy. MN-G-NPs were characterized for shape, particle size, zeta potential, and percentage drug entrapment. The size of nanoparticles was found to be in range of 248-325 nm, and maximum drug payload was found to be 40.2% to 48.5%. Average size was found to be more, but drug payload was less in the case of MN-G-NPs as compared with unconjugated nanoparticles (G-NPs). The results of the in vitro release profile demonstrated that G-NPs release a comparatively higher percentage of drug than MN-G-NPs. Cellular uptake by MN-G-NPs was 2.7 times more as compared with G-NPs. Fluorescence studies revealed the enhanced uptake of MN-G-NPs in the macrophage tissues when compared with unmodified G-NPs. Intravenous administration of free-drug solution resulted in a high concentration of drug in serum, whereas it was much less in the case of G-NPs. Coupling of the nanoparticles with mannose significantly enhanced the lung, liver, and lymph nodes uptake of drug, which is reflected in the recovery of a higher percentage of the dose from these organs following administration of MN-G-NPs in comparison to noncoupled G-NPs or free drug.     

Long-Circulating Poly(Ethylene Glycol)-Modified Gelatin Nanoparticles for Intracellular Delivery

Purpose. The objective of this study was to develop and characterize long-circulating, biodegradable, and biocompatible nanoparticulate formulation as an intracellular delivery vehicle. Methods. Poly(ethylene glycol) (PEG)-modified gelatin was synthesized by reacting Type-B gelatin with PEG-epoxide. The nanoparticles, prepared by pH and temperature controlled ethanol-water solvent displacement technique, were characterized for mean size, size distribution, and surface morphology. Electron spectroscopy for chemical analysis (ESCA) was used to confirm the surface presence of PEG chains. In vitro release of tetramethylrhodamine-labeled dextran (TMR-dextran, Mol. wt. 10,000 daltons) from the nanoparticle formulations was examined in PBS, with and without 0.2-mg/ml protease, at 37°C. Relative cytotoxicity profile of control and PEGylated gelatin was evaluated in BT-20 a human breast cancer cell line. The nanoparticles were incubated with BT-20 cells to determine uptake and cellular distribution using confocal microscopy. Results. Gelatin and PEGylated gelatin nanoparticles were found to be spherical in shape with a smooth surface in a size range of 200-500 nm and a unimodal size distribution. ESCA results showed an increase in the ether carbon (-C-O-) peak in the PEGylated gelatin nanoparticles due to the presence of PEG chains. The presence of PEG chains decreased the percent release of TMR-dextran in the presence of proteolytic enzyme due to steric repulsion. Cytotoxicity assays indicated that both gelatin and PEGylated gelatin were completely non-toxic to the cells. A large fraction of the administered control gelatin and PEGylated gelatin nanoparticles were found to be concentrated in the perinuclear region of the BT-20 cells after 12 hours indicating possible vesicular transport through initial uptake by endocytosis and endosomal processing. Conclusion. The results of this study show that PEGylation of gelatin may prove beneficial as long-circulating delivery system in vivo. Additionally, the nanoparticles could encapsulate hydrophilic macromolecules and are internalized by tumor cells.     

Alive attenuated Salmonella as a cargo shuttle for smart carrying of gold nanoparticles to tumour hypoxic regions

Abstract

In the present study, alive attenuated Salmonella typhi Ty21a was introduced as a vehicle for smart delivery of gold nanoparticles to the tumours’ hypoxic regions. At the first step, the uptakes of gold nanoparticles with seven different decorations by S. typhi Ty21a was investigated using flow cytometry and inductively coupled plasma optical emission spectroscopy. The analyses demonstrated that folic acid functionalised gold nanoparticles (FA-GNPs) are the best candidates for producing the Golden Bacteria (GB). Subsequently, the GB and FA-GNPs efficacies for tumour targeting were investigated after intravenous injection to CT-26 tumour-bearing mice. The GB exhibited more GNPs delivery to the tumour in comparison with FA-GNPs. Moreover, GB injection causes more delivery of GNPs to the tumours’ central regions in comparison with tumours’ periphery. This trend is completely in reverse for FA-GNPs injected group. The ratios of peripheral to central regions’ gold concentration of the tumours were 1.95?±?0.13 and 0.61?±?0.10 for FA-GNPs and GB groups, respectively. This observation demonstrates higher accumulation of gold nanoparticles in the centre of the tumour due to their active delivery by the S. typhi Ty21a to the deeps of tumours.     

Nanoparticulate systems for the delivery of antisense oligonucleotides

Antisense oligonucleotides are molecules that are able to inhibit gene expression being therefore potentially active for the treatment of viral infections or cancer. However, because of their poor stability in biological medium and their weak intracellular penetration, colloidal drugs carriers such as nanoparticles were developed for the delivery of oligonucleotides (ODN). ODN associated to nanoparticles were shown to be protected against degradation and to penetrate more easily into different types of cells. As a consequence, nanoparticles were shown to improve the efficiency of ODNs for the inhibition of the proliferation of cells expressing the point mutated Ha-ras gene. In vivo, polyalkylcyanoacrylate (PACA) nanoparticles were able to efficiently distribute the ODNs to the liver whereas the alginate nanosponges could concentrate the ODNs in the lungs. Finally, ODN loaded to PACA nanoparticles were able to improve in mice, the treatment of RAS cells expressing the point mutated Ha-ras gene.     

Encapsulation of Fe3O4 in gelatin nanoparticles: effect of different parameters on size and stability of the colloidal dispersion

Encapsulation of magnetite (IOPs) in gelatin nanoparticles has been carried out by in situ precipitation of the particles in presence of gelatin, followed by desolvation and cross-linking of the composite nanoparticles. The aim of the study was to investigate the effect of various formulation parameters (viz; desolvating agent, cross-linking agent and percentage of IOPs) on the hydrodynamic size of the gelatin-coated magnetic iron oxide composite nanoparticles (GIOPs) and stability of the colloidal dispersion. Extensive characterization by dynamic light scattering, thermogravimetric analysis, X-ray diffraction, infrared spectroscopy, transmission electron microscopy and atomic force microscopy shows complete encapsulation of IOPs of size below 8 nm into gelatin nanoparticles of varying size. Size as well as stability of the colloidal dispersion of the GIOPs was found to be dependent on the investigated parameters. Furthermore, the nanoparticle dispersion was found to be stable in pH ranges from 2-12. Thus, obtained composite nanoparticles could hold promise as a carrier system in biomedical applications.     

Poly(ethylene glycol)-modified thiolated gelatin nanoparticles for glutathione-responsive intracellular DNA delivery

Poly(ethylene glycol) (PEG)-modified thiolated gelatin (PEG-SHGel) anoparticles were developed as a long-circulating passively targeted delivery system that responds to intracellular glutathione concentrations to enhance DNA delivery and transfection. Reporter plasmid expressing enhanced green fluorescent protein (EGFP-N1) was encapsulated in the nanoparticles. DNA-containing gelatin (Gel) and thiolated gelatin (SHGel) nanoparticles were found to have a size range of 220 to 250 nm, whereas surface modification with PEG resulted in particles with a slightly larger size range of 310 to 350 nm. PEG modification was confirmed by electron spectroscopy for chemical analysis (ESCA), where an increase in the ether peak intensities of the C1s spectra corresponds to the surface presence of ethylene oxide residues. In addition, the PEG-SHGel nanoparticles released encapsulate plasmid DNA in response to varying concentrations of glutathione (up to 5.0 mM GSH in phosphate-buffered saline, or PBS). The stability of the encapsulated DNA was confirmed by agarose gel electrophoresis. Finally, from the qualitative and quantitative results of in vitro transfection studies in murine fibroblast cells (NIH3T3), PEG-Gel and PEG-SHGel nanoparticles afforded the highest transfection efficiency of the reporter plasmid. The results of these studies show that PEG-modified thiolated gelatin nanoparticles could serve as a very efficient nanoparticulate vector for systemic DNA delivery to solid tumors where the cells are known to have significantly higher intracellular GSH concentrations.     

Topical Application of Keratinocyte Growth Factor Conjugated Gold Nanoparticles Accelerate Wound Healing

Keratinocyte growth factor (KGF) has been demonstrated to specifically stimulate the multiplication and migration of keratinocytes. However, due to rapid degradation, the results of topical application of growth factors on wounds are unsatisfactory. In this study, we cross-linked KGF to the surface of gold nanoparticles (GNPs) and explored their effects on wound healing. The as-synthesized nanocomposite (KGF-GNPs) displayed good colloidal stability, decent biocompatibility as well as negligible cellular cytotoxicity. The in vitro cellular experimental results demonstrated that KGF-GNPs could effectively promote the proliferation of keratinocytes in contrast to bare GNPs or KGF. Furthermore, in animal full-thickness wound model, KGF-GNPs are more conducive to wound healing than bare GNPs or KGF. KGF-GNPs enhanced wound healing by promoting wound re-epithelialization rather than granulation. The superior biocompatibility, colloidal depressiveness and biological activity of this nanocomposite indicate that it could be utilized as a promising wound healing drug for clinical application in the future.     

Interaction of gold nanoparticles and nickel(II) sulfate affects dendritic cell maturation

Despite many investigations have focused on the pristine toxicity of gold nanoparticles (GNPs), little is known about the outcome of co-exposure and interaction of GNPs with heavy metals which can possibly detoxify or potentiate them. Here, the combined exposure of nickel (II) sulfate (NiSO₄) and GNPs on the maturation response of dendritic cells (DCs) was explored. Exposure to GNPs or NiSO₄ separately induced cell activation. When cells were exposed to a mixture of both, however, the observed cell activation pattern indicated a competitive rather than an additive effect of both inducers with levels similar to those induced by NiSO₄ alone. Quantification of the GNP uptake by DCs demonstrated a significant decrease in intracellular gold content during co-incubation with NiSO₄. An extensive physiochemical characterization was performed to determine the interaction between GNPs and NiSO₄ in the complex physiological media using nanoparticle tracking analyses, disc centrifugation, UV–visible spectroscopy, ICP-MS analyses, zeta potential measurements, electron microscopy, and proteomics. Although GNPs and NiSO₄ did not directly interact with each other, the presence of NiSO₄ in the physiological media resulted in changes in GNPs' charge and their associated protein corona (content and composition), which may contribute to a decreased cellular uptake of GNPs and sustaining the nickel-induced DC maturation. The presented results provide new insights in the interaction of heavy metals and NPs in complex physiological media. Moreover, this study highlights the necessity of mixture toxicology, since these combined exposures are highly relevant for human subjection to NPs and risk assessment of nanomaterials.     

Development, characterization, and toxicity evaluation of amphotericin B-loaded gelatin nanoparticles

Our aim in the present investigation was to develop a nanoparticulate carrier of amphotericin B (AmB) for controlled delivery as well as reduced toxicity. Nanoparticles of different gelatins (GNPs) (type A or B) were prepared by two-step desolvation method and optimized for temperature, pH, amount of cross-linker, and theoretical drug loading. AmB-loaded GNPs were characterized for size, polydispersity index (PI), shape, morphology, surface charge, drug release, and hemolysis. The developed GNPs (GNP(A300)) were found to be of nanometric size (213 +/- 10 nm), having low PI (0.092 +/- 0.015) and good entrapment efficiency (49.0 +/- 2.9%). All GNPs showed biphasic release characterized by an initial burst followed by controlled release. The in vivo hematological toxicity results suggest nonsignificant reduction (P > .05) in hemoglobin concentration and hematocrit. Nephrotoxicity results showed that there was a nonsignificant (P > .05) increase in blood urea nitrogen and serum creatinine levels. The results confirm that developed GNPs could optimize AmB delivery in terms of cost and safety, and type A gelatin with bloom number 300 was found suitable for such preparation.