Biodistribution and Toxicity Assessment of Superparamagnetic Iron Oxide Nanoparticles In Vitro and In Vivo

Biodistribution and toxicity assessment are critical for safe clinical use of newly developed medicines.Superparamagnetic iron oxide nanoparticles (SPION)are effective carriers for targeted drug delivery.This study aimed to examine the toxicity and biodistribution of SPION coated with polyethylenimine (PEI)(SPION-PEI)designed for small interfering RNA (siRNA) delivery both in vitro and in vivo.SPION-PEI/siRNA complexes were prepared at different weight ratios.Cytotoxic effects of SPION-PEI/siRNA on HSC-T6 cell viability were determined by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).Rats were divided into three groups:a control group,a normal-saline group and a SPION-PEI/siRNA group.After a single intravenous injection,in vivo nanoparticle biodistribution and accumulation were evaluated by Prussian blue staining in the heart,liver,spleen,lung and kidney 8 h,24 h,and 7 days after the injection.Their distribution was histologically studied at the three time points by measuring ironpositive areas (μm2)in organ sections stained with Prussian blue.The same organs were analyzed by H&E staining for any possible histopathological changes.Furthermore,biochemical indexes such as alanine amino transaminase (ALT),aspartate transaminase (AST),blood urea nitrogen (BUN)and creatinine (CREA)were also assessed at all experimental time points.Electrophoresis exhibited that the SPION-PEI could retard siRNA altogether at weight ratios above 4.MTT assay showed that SPION-PEI loaded with siRNA had low cytotoxicity.In vivo study revealed that the liver and spleen were the major sites of SPION-PEI/siRNA deposition.The iron content was significantly increased in the liver and spleen,peaking 24 h after intravenous injection and then declining gradually.No evidence was found of irreversible histopathological damage to any of the organs tested.These results suggested that most SPION-PEI/siRNA complexes were distributed in the liver and spleen,which might be the target organs of SPION-PEI/siRNA complexes.SPION- PEI/siRNA may serve as in vivo carrier for biomedical medicines.

A Novel Technique for the Preparation of ^(125)I-5-trimethylstannyl-1-(2-deoxy-2-fluoro-beta-D-arabino-furanosyl) Urail and Its Biodistribution Pattern in Kunming Mice

In this study, a novel technique for the preparation of 125I-5-trimethylstannyl-1-（2-deoxy-2-fluoro-beta-D-arabinofuranosyl） urail （FIAU） was developed, 125I-FIAU biodistribution profile was detected in Kunming mice and the possibility of using FTAU radio-labeling for reporter gene imaging was explored. 5-trimethylstannyl-1-（2-deoxy-2-fluoro-beta-D-arabinofuranosyl） urail （FTAU） was labeled with radioiodine （125I）. A rotary evaporation method was used to remove excess methanol. The reactant was purified through a Sep-Pak C18 reversal phase column. The radiochemical purity and in vivo stability were determined using silica gel thin layer chromatography （TLC）. The biodistribution of 125I-FIAU in Kunming mice was also detected. The results showed that 125I-FIAU could be radiolabeled effectively with FTAU, with mean labeling rate being （81±0.38）% （n =5）. The mean radiochemical purity of （98.01±0.40）% （n=5） was achieved after a reversal phase Sep-park column purification. 125I-FIAU was stable when incubated in normal human serum or in saline at 37°C, with a radiochemical purity 〉96% during a 0.5-24 h time period. Biological experiments exhibited rapid clearance of 125I-FIAU from the blood pool. 125I-FIAU was mostly excreted by kidneys. 125I-FIAU in myocardium dropped conspicuously after 8 h and there was hardly retention at 24 h. We were led to concluded that the new method of radioiodinization of FTAU for the preparation of 125I-FIAU is easy, highly effective and stable in vivo. The biodistribution of 125I-FIAU in Kunming mice showed it can serve as an imaging probe for myocardial reporter genes.

In vivo biodistribution of topical low molecular weight heparin-taurocholate in a neovascularized mouse cornea

AIM： To investigate the ocular biodistribution and clearance of topically administered 7-taurocholic acid conjugated low-molecular weight heparin（LHT7） in a neovascularized mouse cornea using an in vivo optical imaging system. METHODS： A total of 10 eyes of 6 to 8-week-old BALB/c mice were analyzed. Corneal neovascularization（CoNV） was induced in the inferior cornea（IC） of each animal by penetrating the stroma with two interrupted sutures. The development of CoNV was verified after one week and the area of each neovascularized region was measured. A near-infrared fluorescent probe of 20 μmol/L Cy5.5 labeled LHT7（LHT7-Cy5.5） in 0.02 mL solution was topically instilled onto the cornea in the experimental group（n=5）. Free-Cy5.5 of 20 μmol/L in 0.02 mL was instilled in the control group（n=5）. In vivo optical images were obtained before instillation and 5 min, 2, 4, and 6 h after instillation. The intensities were separately measured at the superior cornea（SC） and the IC. RESULTS： The mean CoNV areas were 1.97±0.17 mm^2 and 1.92±0.96 mm^2 in the experimental and control groups, respectively（P=0.832）. The SC remained normal in all 10 subject animals. The IC intensity of the LHT7-Cy5.5 was greater than the SC intensity at 5 min（P=0.038）, 2 h（P=0.041）, and 4 h（P=0.041） after application. The IC intensity fell to less than half of its initial value（42.9%±8.6%） at 6 h in the experimental group. In the control mice, here were no significant differences in the free-Cy5.5 intensity between the IC and SC. CONCLUSION： Topically administered LHT7 shows a high biodistribution in CoNV areas for 4 h and should be reapplied accordingly to maintain its effects. In vivo optical imaging can be a useful tool for evaluating the ocular biodistribution of a drug in an animal model.

The biodistribution and kinetics of the Samarium-153 labeled avidin,streptavidin and biotin

Objective:To labelavidin(Av)or streptavidin(SA)with 153 Sm by takingadvantageof thehighbindingaffin-ityof biotinto Av or SA.Methods:A biotinderivative(DTPA-biotin)wasradiolabelledwith 153 Sm andthenboundto Av or SA.Thein vivo kineticsandbiodistributionof 153 Sm-labeledAv,SA andDTPA-biotinwerestudiedinratsandmice.Results:153 Sm-Avwascharacterizedby rapidclearancefromthebloodwithhighliverandrenaluptake;153 Sm-SAwas clearedfromthebloodslowlywithhighretentionintheliver,spleenandkidney,whereas 153 Sm-DTPA-biotinmetabolismwas accelerated,anditsexcretionwasmainlythroughthekidney.Conclu sion:Thebiodistributiondifferenceof SAandAvmay providean experimentalbasisfor theselectionof differentcomponentsof avidin-biotinsystemin pretagetingradioim-munoimagingandradioimmunotherapy.

Development of novel interferon alpha2b muteins and study the pharmacokinetic and biodistribution profiles in animal model

Novel human interferon alpha 2b (hIFNα2b) muteins were developed by substituting cysteine residue (C) at positions 2 and 99 with aspartic acid residues (D). The mutein forms were then studied for pharmacokinetic profile. In addition, the influence of charge on the protein structure was tested in vivo for the biodistribution pattern. Codon substitutions were performed by Polymerase Chain Reaction (PCR)-based site-directed mutagenesis on a previously constructed synthetic hIFNα2b open reading frame (ORF) cloned in pET32b expression plasmid. The result of nucleotide sequencing analysis confirmed that all codons were replaced successfully without any additional mutation. Three mutant forms of hIFNα2b ORF were overexpressed in Escherichia coli BL21 (DE3) resulted in three muteins: hIFNα2b C2D, hIFNα2b C99D, hIFNα2b C2D C99D. To follow the kinetic and localization of the mutein interferon after intravenous administration, Tc99m was used to label the proteins. In particular of elimination half-life, it was shown that hIFNα2b C2D C99D > hIFNα2bC2D > hIFNα2bC99D > wild type. hIFNα2b C2D C99D mutein showed highest blood accumulation after 30 minutes administration. Taken together, the charge of hIFNα2b seems to be responsible for the fate of hIFNα2b in vivo.

Biodistribution of fullerene derivative C_(60)(OH)_x(O)_y

The biological behavior of fullerene derivatives shows their considerable potential application in medicine field. To understand the biodistribution of C60 derivatives as drugs or drug carriers, a simple water-soluble derivative C60(OH)x(O)y was labeled with 99mTc. A r-counter and a single photo emission computed tomography (SPECT) were used to assess the biodistribution and metabolism of the labeled compound in mice and rabbits, respectively. The results showed that the compound could be absorbed rapidly by tissues, especially by coronal bone, breastbone, backbone, extremity honeycomb, liver and spleen. The clearance was slow from all tissues except for brain. The compound might be excreted through urine and enteron. The biodistribution shows some difference from that of Yamago et al. In this note, we discuss the possible reason leading to the difference. Further study is needed to find out to what extent the C60 itself determines the biodistribution of derivatives.

Preliminary radioimmunoimaging and biodistribution of ^131iodine-labeled single-chain antibody fragment against progastrin-releasing peptide（31-98） in small cell lung cancer xenografts

Background Monoclonal antibodies （mAbs） such as DD3,raised against progastrin-releasing peptide（31-98） （ProGRP（31-98）） antigen,have been used to target small cell lung cancer （SCLC）.However,as an intact mAb,DD3 is cleared slowly from the body,with an optimal radioimmunoimaging time of 72 hours.More recently,a singlechain antibody fragment has demonstrated reduced excretion time in blood and normal tissues and is increasingly used in diagnostic cancer research.Thereby,it potentially increases the radioimmunoimaging efficacy.However,there have been few studies with this antibody fragment.The aim of this study was to characterize the preliminary radioimmunoimaging and biodistribution of 1311I-anti-ProGRP（31-98）scFv in nude mice bearing SCLC xenografts.Methods Anti-ProGRP（31-98） scFv was used to detect ProGRP expression by flow cytometry analysis and immunohistochemistry.131I-anti-ProGRP（31-98） scFv was injected intravenously into healthy Kunming mice and the percentage injected dose per gram （%ID/g） in various organs was calculated.Similarly,the %ID/g and tumor/non-tumor ratio in xenograft-bearing mice was calculated.After injection of 131I-anti-ProGRP（31-98） scFv,treated mice were imaged at 1,24,and 30 hours.Then the tumor/base ratios were calculated.Results ProGRP was highly expressed in NCI-H446 cells and xenograft tissue.The metabolism of 131I-anti-ProGRP（31-98） scFv in healthy mice was consistent with a first-order and two-compartment model; T1/2α and T1/2β were 10.2 minutes and 5 hours 18 minutes,respectively.The %ID/g of 131I-anti-ProGRP（31-98） scFv in xenografts was much higher than in healthy tissues at 12 hours after injection,reaching a maximum of （5.38±0.92） %ID/g at 24 hours.Successful imaging of xenograft tissue was achieved as early as 1 hour post-injection and persisted until 30 hours,with 24 hours proving optimal.Conclusion 131I-anti-ProGRP（31-98）scFv shows highly selective tumor uptake with low accumulation in normal tissues and rapid blood clearance,indicating thatit could be a promising agent for SCLC radioimmunoimaging.

Radiolabeling LyP-1 peptide and preliminary biodistribution evaluation in mice bearing MDA-MB-435 xenografts

Background Recent studies have shown the LyP-1 peptide can home to either tumor lymphatics or the tumor cells and be internalized by targeted cells. This study aimed to investigate the possibility of using Na1311 labeled LyP-1 peptide as an imaging agent or a therapeutic radiopharmaceutical in breast carcinoma and its metastasis. Methods The 10-mer cyclic peptide contained the LyP-1 sequence （YCGNKRTRGC） was synthesized by the solid phase method. Disulfide bonds between the cysteines maintain the cyclic structure. The LyP-1 peptide was labeled with Na1311 using the chloramine-T method. The [1311] LyP-1 peptide and a [1311] control peptide were injected via tail vein into nude mice bearing MDA-MB-435 tumor xenografts. Biodistribution and imaging results in vivo were obtained. Results The labeling efficiencies of LyP-1 peptide reached 80%＋5% （n=5）. The radiochemical purity was about 96%. The radiochemical purity of the labeled compound remains 92% at 24 hours in human serum at 37~C. In the biodistribution studies, the [1311] LyP-1 peptide accumulated in the tumor to a higher level than in other organs. The [1311] LyP-1 peptide can successfully image the tumor in nude mice bearing MDA-MB-435 tumor xenografts. Conclusions The LyP-1 peptide could be effectively labeled with Na1311 and the labeled compound is stable in human serum at 37℃ for 24 hours. The high specificity of [1311] LyP-1 peptide suggests it may be a promising new radiotracer for identifying tumors.

Preparation of a New Radiolabeled Biomaterial and Its Biodistribution in Mice

Biomaterials have attracted more attention from biomedical research in recent years. Yet there are still unmet demands for current biomaterials, such as the reduction of local inflammation of the implantation site. Poly-Propylene Carbonate （PPC）, a polymer with ester bonds on COz backbone, degrades to CO2 and water, which are natural components of human body, yielding less inflammatory response than traditional biomaterials. However, the tensile strength and heat resistance properties of PPC are less ideal. In order to improve the properties of PPC, we have developed a new PPC （M-PPC）, modified by mixing with Poly-3-Hydroxybutyrate （PHB）. Here, we report the biodistribution profiles of PPC and M-PPC, their biocompatibility and toxicity. 125I-radiolabeled PPC and M-PPC were prepared and their biodistribution in Balb/c mice were investigated. Then acute systemic toxicity and haemolysis assays were conducted to study their toxicity and biocompatibility respectively. Results show that M-PPC has a good potential to be used as bone repair materials because it possesses typical biodistribution pattern in major organs, minimal toxicity and good biocompatibility.

Synthesis of N-(2-^(18)F-fluoropropionly)-Lglutamine and its biodistribution study in mice

Objective To synthesize18F labeled N-(2-18F-fluoropropionyl)-L-glutamine(18F-FPGLN)for tumor PET imaging,and to perform its biodistribution study on normal mice and PC-3 tumorbearing nude mice.Methods4-nitrophenyl-2-18F-fluropropionate(18F-NFP)was synthesized on the MF-2V-IT-I synthesizer and was

Liposomes for systematic delivery of vancomycin hydrochloride to decrease nephrotoxicity:Characterization and evaluation

Vancomycin hydrochloride(VANH),the first glycopeptide antibiotic,is a water-soluble drug for the treatment of acute osteomyelitis.Liposomal formulations of VANH have already been manipulated and characterized,which was a mean of increasing their therapeutic index,reducing their toxicity and altering drug biodistribution.One of the challenges for preparing VANH-Lips is their low encapsulation efficiency(EE).In the present study,we aim to improve the liposomal formulation of VANH for higher EE,longer systemic circulation,reduced nephrotoxicity and enhanced antimicrobial activities.Vancomycin hydrochloride-loaded liposomes(VANH-Lips)were formulated by the method of modified reverse phase evaporation.Based on the optimization of formulation with orthogonal experimental design,the average drug encapsulation efficiency and the mean particle size of VANH-Lips were found to be 40.78±2.56%and 188.4±2.77 nm.In vitro drug release of VANH-Lips possessed a sustained release characteristic and their release behavior was in accordance with the Weibull equation.After intravenous injection to mice,the mean residence time(MRT)of VANH-Lips group was significantly prolonged in vivo and the AUC value was improved as well compared with the vancomycin hydrochloride solution(VANH-Sol)group.Furthermore,the biodistribution results in mice showed that VANH-Lips decreased the accumulation of VANH in kidney after intravenous injection.In conclusion,VANH-Lips may be a potential delivery system for VANH to decrease nephrotoxicity in the treatment of osteomyelitis.

Aspects of high-performance and bio-acceptable magnetic nanoparticles for biomedical application

This review covers extensively the synthesis&surface modification,characterization,and application of magnetic nanoparticles.For biomedical applications,consideration should be given to factors such as design strategies,the synthesis process,coating,and surface passivation.The synthesis method regulates post-synthetic change and specific applications in vitro and in vivo imaging/diagnosis and pharmacotherapy/administration.Special insights have been provided on biodistribution,pharmacokinetics,and toxicity in a living system,which is imperative for their wider application in biology.These nanoparticles can be decorated with multiple contrast agents and thus can also be used as a probe for multi-mode imaging or double/triple imaging,for example,MRI-CT,MRI-PET.Similarly loading with different drug molecules/dye/fluorescent molecules and integration with other carriers have found application not only in locating these particles in vivo but simultaneously target drug delivery/hyperthermia inside the body.Studies are underway to collect the potential of these magnetically driven nanoparticles in various scientific fields such as particle interaction,heat conduction,imaging,and magnetism.Surely,this comprehensive data will help in the further development of advanced techniques for theranostics based on high-performance magnetic nanoparticles and will lead this research area in a new sustainable direction.

Studies on Hepatocyte-Targeting Magnetic Resonance Imaging Macromolecular Contrast Media

DTPA was covalently conjugated to the copolymer of L lysine and L tyrosine, then transchelated with Gd EDTA,the resultant polymer chelates were further reacted with 6 O bromoacetyl D galactose or methyl lactobionyloxyacetate to give PLT(Gd DTPA) with galactose moiety as liver targeting group.These macromolecular MRI contrast agents were characterized by means of FT IR, ICP AES, elementary analyses and UV Vis spectrophotometry. Their in vitro relaxivity and liver targeting property in mice were also investigated.

Preliminary evaluation of the radiotherapeutic efficacy of 131I-atorvastatin in rats with hepatocellular carcinoma

As one of the most critical types of cancer,hepatocellular carcinoma(HCC)affects many people worldwide.This study demonstrated the prospective use of atorvastatin,a drug that inhibits the mevalonate pathway,causing hypolipidemia,as a carrier to deliver the iodine-131(131I)isotope to liver tissues for HCC radiotherapy.The atorvastatin radioiodination method was optimized for utilizing the131I isotope.The radiochemical quality and the in vitro stability of the generated[131I]atorvastatin were investigated.In addition,the biodistribution experiments of[131I]atorvastatin were evaluated in both normal and HCC-induced rat models.[131I]atorvastatin was produced at a maximum radiochemical yield of 86.7±0.49%.The[131I]atorvastatin solution purified via high-performance liquid chromatography showed good in vitro stability for 12 h after tagging.Biodistribution analyses revealed remarkable liver targeting capacity of[131I]atorvastatin and good localization of131I in liver tissues.Overall,the encouraging biochemical profile and histopathologicalfindings have been reported,and preliminary investigations on the possible use of[131I]atorvastatin as a radiotracer and its impact on HCC radiotherapy in rats show promise.

LABELLING OF METALLOTHIONEIN WITH ^(99m)Tc

99mTc-labelled metallothionein (99mTc-MT) was prepared through both direct and transcomplexation labelling approaches. Buffer systems and a variety of other parameters in the direct labelling procedure were studied in detail. High radiolabelling yields of 93-96% and the specific radioactivity of 3.7 MB/μg were obtained under optimal conditions by the direct labelling method. The prepared 99mTc-MT was stable in vitro. The chelate-exchange kinetics of MT with 99mTc-glucohepatonate and 99mTc-citrate were also studied. Biodistribution and imaging studies showed that 99mTc-MT was accumulated in the animal kidneys at an exceptionally high level, indicating that 99mTc-MT might be a potential renal imaging agent.

INTERNAL RADIOTHERAPY OF LIVER CANCER USING^(131)ILIPIODOL

Internal radiotherapy with hepatic artery injection(HAI)and B-ultrasound guidedpuncture injection(BGPI)of 131I-lipiodol was observed in 20 patients with liver cancer.The radinuclide images showed that 131I-lipiodol accumulated in tumor mainly.The radioactivity of tumor/liverratio(T/L)in HAI and BGPI was 3.87±2.77 and 9.15±3.41 resistively. The effective half-lifeof,131I-lipiodol in tumor was(4.5±1.92)anys and(6.22±0.61) days. The radioactivity of lung was(13.19±3.01)% and(5.19±2.28)% espectively.These results suggest that the 131I-lipiodol biodistribution in BGPI is better than that in HAI.Clinical results showed that ABP levels dropped rapidlyin 64.3% of patients with peltive AFP.A shrinkage in tumor size was observed in 66.7% of patients.Besides transient fever,elevated bilirubin and SGPT,no any toxicity and adverse reaction wasobserved.

Biodegradable Guar Gum Nanoparticles as Carrier for Tamoxifen Citrate in Treatment of Breast Cancer

We prepared, characterized and studied the biodistribution of tamoxifen citrate (TMX) loaded cross-linked guar gum (GG) nanoparticles (NPs). NPs were prepared via a single step emulsion process and particle size evaluated. The extent of tissue distribution and retention following oral administration of TMX loaded GG NPs and TMX tablet in female albino mice was analyzed over a period of 48 hours. Till 48 hours, the particles remained detectable in both mammary and ovary tissue (estrogen receptors). Uptake and retention of TMX from NPs and tablet in mammary gland and ovary tissue changed with time. Results showed that the uptake and retention of NPs was more in the mammary gland between 24 - 48 hours (11.2% at 24 h;4.65% at 48 h). As mammary gland is the target organ in breast cancer therapy, it may be concluded that the cross-linked GG NPs are capable of releasing the drug at the target and minimize the uptake and retention in non target tissue, the ovary (7.98% at 24 h;1.9% at 48 h). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) with time were measured. No abnormal changes in the liver enzymes were observed. GG NPs under study can be used as a drug carrier system for treating cancer.

<i>In vivo</i>Distribution of Inorganic Nanoparticles in Preclinical Models

Ongoing progress in nanotechnologies has led to their implementation for in vivo diagnostic and therapy. Thus, the main applications of inorganic nanoparticles are imaging for diagnosis and cell tracking, photothermal and drug-delivery therapies. Following nanoparticles in vivo administration, the systemic circulation can distribute them to every body organ and tissue. Precise characterization of nanoparticles distribution and accumulation in the different body parts in preclinical models is required before any application in humans. The biodistribution of inorganic nanoparticles has been analysed in different preclinical models, particularly mouse, rat and rabbit. This review covers the in vivo biodistribution of different inorganic nanoparticles in preclinical models: gold nanoparticles, silica nanoparticles, iron oxide magnetic nanoparticles, quantum dots and carbon nanotubes.

Synthesis and Radiation Dosimetry of [⁶⁸Ga]-Ga-Lys¹, Lys³-DOTA-Bombesin (1,14) Antagonist for PET-Imaging, as a Potential Theragnostic Tracer in Oncology

This Bombesin (BBN), a tetradecapeptide analog of human gastrin-releasing peptide (GRP) with a high binding affinity for GRP receptors (GRPR), is over- expressed in early stages of androgen-dependent prostate carcinomas, but not in advanced stages. Therefore, there is a need to develop effective tracers for the accurate and specific detection of this disease. The objective of this study was to evaluate Lys1, Lys3-DOTA-BBN (1,14) analog with the radiolabeled positron emitter [68Ga]-Ga-BBN for receptor imaging with PET, and to determine its biodistribution and radiation dosimetry using whole-body (WB) PET scans in healthy volunteers. The highest uptake was in the pancreas, followed by urinary bladder. The critical organ was pancreas with a mean absorbed dose of 206 ± 0.7, 210 ± 0.7, 120 ± 0.9, 390.23 ± 0.6 μGy/MBq and the effective doses were estimated as 73.2 ± 0.6, 49.8 ± 0.3 μGy/MBq (women and men, respectively).

Oral peptide therapeutics for diabetes treatment: State-of-the-art and future perspectives

Diabetes,characterized by hyperglycemia,is a major cause of death and disability worldwide.Peptides,such as insulin and glucagon-like peptide-1(GLP-1)analogs,have shown promise as treatments for diabetes due to their ability to mimic or enhance insulin's actions in the body.Compared to subcutaneous injection,oral administration of anti-diabetic peptides is a preferred approach.However,biological barriers significantly reduce the efficacy of oral peptide therapeutics.Recent advancements in drug delivery systems and formulation techniques have greatly improved the oral delivery of peptide therapeutics and their efficacy in treating diabetes.This review will highlight(1)the benefits of oral anti-diabetic peptide therapeutics;(2)the biological barriers for oral peptide delivery,including pH and enzyme degradation,intestinal mucosa barrier,and biodistribution barrier;(3)the delivery platforms to overcome these biological barriers.Additionally,the review will discuss the prospects in this field.The information provided in this review will serve as a valuable guide for future developments in oral anti-diabetic peptide therapeutics.