Liver Biodistribution Research Articles

Glycocalyx-Mimicking Nanoparticles with Differential Organ Selectivity for Drug Delivery and Therapy.

Organ-selective drug delivery is expected to maximize the efficacy of various therapeutic modalities while minimizing their systemic toxicity. Lipid nanoparticles and polymersomes can direct the organ-selective delivery of mRNAs or gene editing machineries, but their delivery is limited to mostly liver, spleen, and lung. A platform that enables delivery to these and other target organs is urgently needed. Here, a library of glycocalyx-mimicking nanoparticles (GlyNPs) comprising five randomly combined sugar moieties is generated, and direct in vivo library screening is used to identify GlyNPs with preferential biodistribution in liver, spleen, lung, kidneys, heart, and brain. Each organ-targeting GlyNP hit show cellular tropism within the organ. Liver, kidney, and spleen-targeting GlyNP hits equipped with therapeutics effectively can alleviate the symptoms of acetaminophen-induced liver injury, cisplatin-induced kidney injury, and immune thrombocytopenia in mice, respectively. Furthermore, the differential organ targeting of GlyNP hits is influenced not by the protein corona but by the sugar moieties displayed on their surface. It is envisioned that the GlyNP-based platform may enable the organ- and cell-targeted delivery of therapeutic cargoes.

Lactosylated Chitosan Nanoparticles Potentiate the Anticancer Effects of Telmisartan In Vitro and in a N-Nitrosodiethylamine-Induced Mice Model of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the leading cause of cancer-related mortality worldwide. Telmisartan (TLM), a BSC class II drug, has been reported to have antiproliferative activity in HCC. However, its therapeutic activity is limited by poor bioavailability and unpredictable distribution. This work aimed to enhance TLM's liver uptake for HCC management through passive and active targeting pathways utilizing chitosan nanoparticles decorated with lactose (LCH NPs) as a delivery system. In vitro cell cytotoxicity and cellular uptake studies indicated that TLM-LCH NPs significantly (p < 0.05) enhanced the antiproliferative activity and cellular uptake percentage of TLM. In vivo bioavailability and liver biodistribution studies indicated that TLM-LCH NPs significantly (p < 0.05) enhanced TLM concentrations in plasma and the liver. The relative liver uptake of TLM from TLM-LCH NPs was 2-fold higher than that of unmodified NPs and 5-fold higher than that of plain TLM suspension. In vivo studies of a N-nitrosodiethylamine-induced HCC model revealed that administration of TLM through LCH NPs improved liver histology and resulted in lower serum alpha-fetoprotein (AFP), matrix metalloproteinase 2 (MMP-2), vascular endothelial growth factor (VEGF) levels, and liver weight index compared to plain TLM and TLM-loaded unmodified NPs. These results reflected the high potentiality of LCH NPs as a liver-targeted delivery system for TLM in the treatment of HCC.

Intravenous administration of three multiwalled carbon nanotubes to female rats and their effect on urinary biochemical profile.

This study was conducted to investigate the influence of outer diameter (OD) and length (L) of multiwalled carbon nanotubes (MWCNTs) on biodistribution and the perturbation of endogenous metabolite profiles. Three different-sized carboxylated MWCNTs (NIEHS-12-2: L 0.5-2μm, OD 10-20 nm, NIEHS-13-2: L 0.5-2μm, OD 30-50 nm, and NIEHS-14-2: L 10-30 μm, OD 10-20 nm) in water were administered to female Sprague-Dawley rats as a single intravenous dose of 1mg/kg MWCNTs. Biodistribution in liver, lung, spleen, and lymph nodes was evaluated in tissue sections at 1 and 7days' post-dosing using enhanced darkfield microscopy and hyperspectral imaging. Nuclear magnetic resonance (NMR) analysis was used for biochemical profiling and pathway mapping of endogenous metabolites in urine collected at 24-h intervals prior to dosing, at Day 1 and Day 7. At Day 1 and Day 7, all three MWCNTs were observed in liver. NIEHS-12-2 was observed in spleen, whereas NIEHS-13-2 and NIEHS-14-2 were not. All three MWCNTs were observed in lymph nodes and lung at Day 7. The urinary biochemical profile showed the highest positive fold change (FC) at Day 7 for the metabolites acetate, alanine, and lactate, whereas 1-methylnicotinamide, 2-oxoglutarate, and hippurate had some of the lowest FCs for all three MWCNTs. This study demonstrates that the observed tissue location of MWCNTs is size dependent. Overlaps in the perturbation of endogenous metabolite profiles were found regardless of their size, and the biochemical responses were more profound at Day 7 compared with Day 1, indicating a delayed biological response to MWCNTs.

Live cell imaging of highly activated natural killer cells against human hepatocellular carcinoma in vivo

Background aimsTracking administered natural killer (NK) cells in vivo is critical for developing an effective NK cell-based immunotherapy against human hepatocellular carcinoma (HCC). Here the authors established a new molecular imaging using ex vivo-activated NK cells and investigated real-time biodistribution of administered NK cells during HCC progression. MethodsEx vivo-expanded NK cells from healthy donors were labeled with a near-infrared lipophilic cytoplasmic dye, and their proliferation, surface receptor expression and cytotoxicity activity were evaluated. Human HCC HepG2 cells were implanted into the livers of NOD.Cg-Prkdcscid IL2rgtm1Wjl/SzJ (NSG) mice. The authors administered 1,1’-dioctadecyltetramethyl indotricarbocyanine iodide (DiR)-labeled NK cells intravenously to non-tumor-bearing and intrahepatic HCC tumor-bearing NSG mice. Fluorescent imaging was performed using a fluorescence-labeled organism bioimaging instrument. Single cell suspensions from the resected organs were analyzed using flow cytometry. ResultsThe fluorescent DiR dye was nontoxic and did not affect the proliferation or surface receptor expression levels of the NK cells, even at high doses. The administered DiR-labeled NK cells immediately migrated to the lungs of the non-tumor-bearing NSG mice, with increased NK cell signals evident in the liver and spleen after 4 h. NK cells migrated to the intrahepatic tumor-bearing livers of both early- and late-stage HCC mice within 1 h of injection. In early-stage intrahepatic tumor-bearing mice, the fluorescence signal increased in the liver until 48 h post-injection and decreased 7 days after NK injection. In late-stage HCC, the NK cell fluorescence signal was the highest in the liver for 7 days after NK injection and persisted for 14 days. The purity of long-term persistent CD45+CD56+CD3− NK cells was highest in early- and late-stage HepG2-bearing liver compared with normal liver 2 weeks after NK injection, whereas highest purity was still observed in the lungs of non-tumor-bearing mice. In addition, Ki-67 expression was detected in migrated human NK cells in the liver and lung up to 72 h after administration. With HepG2 tumor progression, NK cells reduced the expression of NKp30 and NKG2D. ConclusionsAdministered NK cells were successfully tracked in vivo by labeling the NK cells with near-infrared DiR dye. Highly expanded, activated NK cells migrated rapidly to the tumor-bearing liver, where they persisted for 14 days after administration, with high purity of CD45+CD56+CD3− NK cells. Liver biodistribution and persistence of administered NK cells showed significantly different accumulation patterns during HCC progression.

Abstract No. 4 Accuracy of scout dose Y90 liver biodistribution for personalized treatment planning of Y90 radioembolization of hepatocellular carcinoma: interim analysis of a prospective clinical trial

To evaluate the accuracy of scout dose Yttrium-90 (Y90) resin microspheres versus technetium-99m MAA (Tc99m-MAA) in predicting the therapeutic dose Y90 liver biodistribution for radioembolization (RE) planning in hepatocellular carcinoma (HCC) patients In a prospective single-arm clinical trial patients with HCC undergoing Y90 RE were recruited. Inclusion criteria included unilobar disease with ≤3 lesions with the largest lesion ≤ 7cm. Each patient underwent mapping angiography study with Tc99m-MAA and MAA SPECT/CT followed by second mapping study using 15 mCi Y90 resin microspheres and Y90 SPECT/CT and PET/CT. Prospective treatment planning with MAA data was performed using partition model with the goal of >200 Gy to the targeted tumor. Prescribed Y90 activity minus previously administered scout activity was administered 3 days after the mapping studies. Same microcatheter type and position were used for all stages of the study in each patient. Sureplan (MIM Software, Cleveland, OH) was used for all dosimetry analyses. Using paired t-test, the tumor:normal (TNR) calculated by MAA SPECT and scout dose Y90 SPECT and PET were compared to that of therapeutic dose Y90 SPECT and PET. First N = 15 patients treated in the clinical trial are included in this interim analysis. Mean TNR by MAA SPECT was 2.33. Mean TNR by scout dose Y90 SPECT and PET were 1.79 and 2.59, respectively. Mean TNR by therapeutic dose Y90 SPECT and PET were 1.75 and 2.65, respectively. MAA TNR was greater than both Y90 scout and therapeutic dose TNRs calculated by SPECT (Ps< 0.001). MAA TNR was statistically similar to both scout dose and therapeutic dose Y90 TNRs calculated by PET (P = 0.33 and P = 0.55, respectively). Y90 Scout and therapeutic dose TNRs calculated by SPECT were similar (P = 0.59) and the same finding was observed for Y90 TNRs calculated by Y90 scout and therapeutic dose PET (P = 0.74). Y90 scout dose is an accurate predictor of therapeutic Y90 dose biodistribution in the liver on both SPECT and PET, respectively. On the other hand, Tc99-MAA TNR was significantly greater than both Y90 scout and therapeutic dose TNRs on SPECT, whereas it was statistically similar to that of PET.

Systemic AAV6-synapsin-GFP administration results in lower liver biodistribution, compared to AAV1&2 and AAV9, with neuronal expression following ultrasound-mediated brain delivery

Non-surgical gene delivery to the brain can be achieved following intravenous injection of viral vectors coupled with transcranial MRI-guided focused ultrasound (MRIgFUS) to temporarily and locally permeabilize the blood–brain barrier. Vector and promoter selection can provide neuronal expression in the brain, while limiting biodistribution and expression in peripheral organs. To date, the biodistribution of adeno-associated viruses (AAVs) within peripheral organs had not been quantified following intravenous injection and MRIgFUS delivery to the brain. We evaluated the quantity of viral DNA from the serotypes AAV9, AAV6, and a mosaic AAV1&2, expressing green fluorescent protein (GFP) under the neuron-specific synapsin promoter (syn). AAVs were administered intravenously during MRIgFUS targeting to the striatum and hippocampus in mice. The syn promoter led to undetectable levels of GFP expression in peripheral organs. In the liver, the biodistribution of AAV9 and AAV1&2 was 12.9- and 4.4-fold higher, respectively, compared to AAV6. The percentage of GFP-positive neurons in the FUS-targeted areas of the brain was comparable for AAV6-syn-GFP and AAV1&2-syn-GFP. In summary, MRIgFUS-mediated gene delivery with AAV6-syn-GFP had lower off-target biodistribution in the liver compared to AAV9 and AAV1&2, while providing neuronal GFP expression in the striatum and hippocampus.

SERPINA1 mRNA as a Treatment for Alpha-1 Antitrypsin Deficiency.

Alpha-1-antitrypsin (AAT) deficiency is a genetic disorder that produces inactive/defective AAT due to mutations in the SERPINA1 gene encoding AAT. This disease is associated with decreased activity of AAT in the lungs and deposition of excessive defective AAT protein in the liver. Currently there is no specific treatment for liver disease associated with AAT deficiency. AAT lung disease is often treated with one of several serum protein replacement products; however, long-term studies of the effectiveness of SerpinA1 replacement therapy are not available, and it does not reduce liver damage in AAT deficiency. mRNA therapy could potentially target both the liver and lungs of AAT deficient patients. AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes were transfected with SERPINA1-encoding mRNA and cell culture media were tested for SerpinA1 expression. Our data demonstrates increased SerpinA1 protein in culture media from treated AAT patient fibroblasts and AAT patient fibroblast-derived hepatocytes. In vivo studies in wild type mice demonstrate SERPINA1 mRNA biodistribution in liver and lungs, as well as SerpinA1 protein expression in these two target organs which are critically affected in AAT deficiency. Taken together, our data suggests that SerpinA1 mRNA therapy has the potential to benefit patients suffering from AAT deficiency.

PEG/Dextran Double Layer Influences Fe Ion Release and Colloidal Stability of Iron Oxide Nanoparticles

Despite preliminary confidence on biosafety of polymer coated iron oxide nanoparticles (SPIONs), toxicity concerns have hampered their clinical translation. SPIONs toxicity is known to be due to catalytic activity of their surface and release of toxic Fe ions originating from the core biodegradation, leading to the generation of reactive oxygen species (ROS). Here, we hypothesized that a double-layer polymeric corona comprising of dextran as an interior, and polyethylene glycol (PEG) as an exterior layer better shields the core SPIONs. We found that ROS generation was cell specific and depended on SPIONs concentration, although it was reduced by sufficient PEG immobilization or 100 µM deferoxamine. 24 h following injection, PEGylated samples showed reduction of biodistribution in liver, heterogenous biodistribution profile in spleen, and no influence on NPs blood retention. Sufficient surface masking or administration of deferoxamine could be beneficial strategies in designing and clinical translation of future biomedical SPIONs.

Triazole- and Tetrazole-Bridged Nucleic Acids: Synthesis, Duplex Stability, Nuclease Resistance, and in Vitro and in Vivo Antisense Potency.

Antisense oligonucleotides are attractive therapeutic agents for several types of disease. One of the most promising modifications of antisense oligonucleotides is the introduction of bridged nucleic acids. As we report here, we designed novel bridged nucleic acids, triazole-bridged nucleic acid (TrNA), and tetrazole-bridged nucleic acid (TeNA), whose sugar conformations are restricted to N-type by heteroaromatic ring-bridged structures. We then successfully synthesized TrNA and TeNA and introduced these monomers into oligonucleotides. In UV-melting experiments, TrNA-modified oligonucleotides exhibited increased binding affinity toward complementary RNA and decreased binding affinity toward complementary DNA, although TeNA-modified oligonucleotides were decomposed under the annealing conditions. Enzymatic degradation experiments demonstrated that introduction of TrNA at the 3'-terminus rendered oligonucleotides resistant to nuclease digestion. Furthermore, we tested the silencing potencies of TrNA-modified antisense oligonucleotides using in vitro and in vivo assays. These experiments revealed that TrNA-modified antisense oligonucleotides induced potent downregulation of gene expression in liver. In addition, TrNA-modified antisense oligonucleotides showed a tendency for increased liver biodistribution. Taken together, our findings indicate that TrNA is a good candidate for practical application in antisense methodology.

Red blood cell membrane camouflaged magnetic nanoclusters for imaging-guided photothermal therapy

Along with intrinsic magnetic resonance imaging (MRI) advantages, iron oxide nanomaterials capable of photothermal conversion have been reported very recently and have again raised great interest in their designs among biomedical researchers. However, like other inorganic nanomaterials, high macrophage uptake, short blood retention time and unfavorable biodistributions have strongly hampered their applications in vivo. To solve these problems, a rational design of red blood cell (RBC) membrane camouflaged iron oxide magnetic clusters (MNC@RBCs) is presented in this paper. Our data show that by simply introducing an “ultra-stealth” biomimetic coating to iron oxide magnetic nanoclusters (MNCs), MNC@RBCs maintain the imaging and photothermal functionalities inherited from MNCs cores while achieving much lower nonspecific macrophage uptake and dramatically altered fate in vivo. MNC@RBCs with superior prolonged blood retention time, preferred high tumor accumulation and relatively lowered liver biodistribution are demonstrated when injected intravenously in mice, leading to greatly enhanced photothermal therapeutic efficacy by a single treatment without further magnetic force manipulation. Our study illustrates a well prepared integration of MNCs and RBCs, exploiting advantages of both functionalities within a single unit and suggests a promising future for iron-based nanomaterials application in vivo.

Bio-inspired polymer envelopes around adenoviral vectors to reduce immunogenicity and improve in vivo kinetics

Adenoviral vectors have attracted substantial interest for systemic tumor gene therapy, but further work is needed to reduce their immunogenicity and alter their biodistribution before they can be used in the clinic. Here we describe a bio-inspired, cleavable PEGylated β-cyclodextrin-polyethyleneimine conjugate (CDPCP) that spontaneously coats adenovirus in solution. This cleavable PEG coating reduces the innate and adaptive immunogenicity of adenovirus particles, as well as improves their biodistribution away from the liver and into the tumor. Insertion of a matrix metalloproteinase substrate sequence into the conjugate allows PEG cleavage at the tumor site, simultaneously reducing liver biodistribution and increasing transgene expression in tumors, thereby avoiding the “PEG dilemma”. Cationic β-cyclodextrin–PEI not only provides electrostatic attraction to promote envelope attachment to the viral capsid, but it also improves vector internalization and transduction after PEG cleavage. These results suggest that CDPCP may help expand the use of adenoviral vectors in cancer gene therapy. Statement of significanceThe synthesized β-cyclodextrin–PEI-MMP-cleavable-PEG polymer (CDPCP), held great potential for gene therapy when applied for adenovirus coating. The β-cyclodextrin–PEI provided a powerful electrostatic attraction to attach the whole polymer onto the viral capsid, while the MMPs-cleavable PEG reduced innate and adaptive immunogenicity and improved the biodistribution of adenovirus vectors due to the tumor-specific enzyme triggered PEG cleavage. More importantly, an ingenious cooperation between the two components could solve the PEG dilemma. The CDPCP/Ad complexes exhibited a comprehensive and valued profile to be a candidate vector for future tumor gene therapy, we believe the current investigation on this kind of biomaterial may be of particular interest to the readership of Acta biomaterialia.

Efeitos do óleo de andiroba (Carapa guianensis) na função hepática de ratos submetidos à isquemia e reperfusão normotérmica do fígado

To evaluate the effects of the Andiroba (carapa guianensis) oil on liver function in rats subjected to normothermic ischemia / reperfusion injury. we divided 12 Wistar rats into two groups: saline (n = 6) and Andiroba (n = 6). The Andiroba group was treated with Andiroba oil (0.63 ml/kg orally) for seven days before surgery. Ischemia was induced by occlusion of the blood supply to the lateral and median lobes of the liver, using vascular clips, in both groups, for 45min, followed by reperfusion for 60 minutes later. We analyzed dosages of AST, ALT, Gamma-GT, and liver biodistribution of 99mTc phytate. There was no significant difference in the percentage of radioactivity / gram of tissue (%ATI/g) in the right lobe of the saline group (17.53 ± 2.78) compared with the Andiroba group (18.04 ± 3.52) p = 0.461, the same occurring in the%ATI/g of the left lobe of the liver when the two groups were compared (p = 0.083). In the saline group, the%ATI/g was significantly higher in the non-ischemic right hepatic lobe (17.53 ± 2.78) when compared with the left lobe (5.04 ± 0.82) that suffered ischemia / reperfusion (p = 0.002). Significant differences also occurred when comparing the right (18.04 ± 3.52) and left (7.11 ± 1.86) lobes of the animals of the Andiroba group (p = 0.004). There was no significant difference in dosages of AST, ALT and Gamma- GT when comparing the two groups (p > 0.05). Andiroba oil did not contribute to the protection of liver function in a rat model of liver injury induced by normothermic ischemia and reperfusion.

Sildenafil in the prevention of hepatic ischemia/reperfusion injury in rats

Purpose: This study aimed to determine the effect of sildenafil on the liver, when administered prior to the episode of liver ischemia/reperfusion. Method: We used 12 Wistar rats. In both IR group (n=6) and IR sildenafil group (n=6), hepatic ischemia was induced for 45 minutes by occlusion of blood vessels supplying the median and lateral lobes of the liver using a vascular clip (bulldog). IR group animals were treated with sildenafil citrate at a dose of 0.7 mg/kg by gavage, 30 minutes before ischemia. During the observation period of ischemia/reperfusion, the abdomen was temporarily closed. After 45 min the vascular clamp was removed, and reperfusion occurred by 60 min. Liver biodistribution of Tc-99m-phytate, histopathology and serum AST, ALT, LDH were analyzed. Results: In animals treated with sildenafil the right lobe of the liver (without ischemia) had significantly higher radioactivity uptake than in the untreated rats (p &lt;0.001). After ischemia/reperfusion, sildenafil failed to improve the radioactive uptake in the left lobe (p = 0.01). Comparing the right and left lobes, the radioactive uptake was lower in lobe left than in right lobe (p = 0.005) of untreated rats (controls). The liver function tests were significantly more impaired in the group treated with sildenafil than in controls. Conclusion: Sildenafil contributed to the deterioration of liver function in a model of ischemia/reperfusion

695. Effect of Neonatal Hepatic Gene Therapy on Cardiovascular Disease, Hearing, and Visual Function in Mice with MPS I

Mucopolysaccharidosis type I (MPS I) is lysosomal storage disorder caused by deficiency of the enzyme, alpha-L-iduronidase (IDUA). We investigated gene therapy for MPS I by using a recombinant lentiviral (HIV-based) vector (pSMPU-MCU3-huID-IRES-eGFP) that contains the normal human IDUA cDNA as well as the enhanced green fluorescence protein (eGFP) reporter gene. We have demonstrated significant levels of IDUA gene expression by this vector in cell lines. To evaluate the effect of age on systemic gene transfer using this vector, we performed a single intravenous injection at different-ages in the IDUA gene knock-out mouse (developed by HH Li and EF Neufeld), treated as neonates (0 or 1-day old) (via facial vein, group 1, n=7), or as young-adults (8-week old) (via tail vein, group 2, n=5), with untreated age-matched control groups. We used the same weight-adjusted dosage of vector (1.65×10e5TU/kg) in neonates and young-adults, and sacrificed them at the same age (20-week old) to investigate the pathological changes, IDUA activity, and vector biodistribution in liver, spleen, kidney, heart, and brain. In the newborn group, almost no lysosomal storage was seen in all 5 organs (there was a little variance in the kidney). In contrast, in the young-adult group there was moderate accumulation in liver and spleen, and high levels were seen in heart, kidney, and brain. The IDUA activity was significantly higher (p<0.05) in the newborn group than in the young-adult group in the spleen (15.2% of normal in newborn group vs. 2.8% in adolescent group), kidney (24.3% vs.4.7%), heart (22.24% vs. 0.3%), and brain (37.7% vs. 1.3%). The higher level of IDUA activity in the liver of mice treated as newborns did not reach statistical significance (27.2% vs. 20.1%). The vector DNA copy numbers determined by real-time PCR for the eGFP sequences in the newborn group were significantly higher only in the brain (0.672% vs. 0.008% genome equivalents/cell) but were relatively higher in the other organs of the newborn group than that of the young-adult group. Expression of the eGFP gene in the brain was mainly in the neurons by immunohistochemistry. In summary, significantly better phenotype correction and vector gene biodistribution were seen in the newborn group compared with the young-adult group, despite the longer time elapsed since gene therapy in the former (20 weeks vs. 12 weeks). We concluded that the systemic injection of lentiviral vector for MPS I at the neonatal stage is much more effective than at the young-adult stage. Possible reasons include the incompletely formed blood-brain barrier, more efficient integration of the vector into neonatal tissues or hematopoietic stem cell, avoidance of deleterious immune response in the neonatal period. For long-term follow-up, we are evaluating the craniofacial skeletal change by x-ray and CT and are dynamically tracking injected vector by using bioluminescence imaging. These studies should be important for the development of gene therapy as an effective treatment for MPS I.

Lymphatic Uptake and Biodistribution of Liposomes after Subcutaneous Injection I. Influence of the Anatomical Site of Injection

Liposomes have been proposed as carriers for the delivery of therapeutic and diagnostic agents to the lymphatic system. Subcutaneous (s.c.) injection is the route of administration most extensively studied for this purpose. To establish the influence of the anatomical site of s.c. injection on lymphatic uptake of liposomes, we have examined the pharmacokinetics and biodistribution of small (0.10 μm) [3H]-labeled liposomes administered at three different s.c. sites of the rat. Lymphatic uptake of liposomes from the s.c. site of injection, lymph node localization and biodistribution in liver, spleen and kidneys were highly dependent on the site of injection. s.c. injection into the dorsal side of the foot or in the footpad resulted in relatively high uptake of liposomes from the site of injection as compared to uptake from the s.c. injection site at the flank. Moreover, injection into the dorsal side of the foot or in the footpad resulted in relatively high levels of radioactivity in regional lymph nodes, liver and spleen. The observed differences in liposome disposition are attributed to differences in the structural organization of s.c. tissue. We conclude that lymphatic uptake and lymph node localization of liposomes after s.c. administration strongly depends on the anatomical site of injection. Therefore, the s.c. site of injection should be carefully considered when developing diagnostic and therapeutic approaches involving the lymphatic system by means of s.c. administration of liposomes.