Macrophage Depletion Reduces Disease Pathology in Factor H-Dependent Immune Complex-Mediated Glomerulonephritis.

Complement factor H (FH) is a key regulator of the alternative pathway of complement, in man and mouse. Earlier, our studies revealed that the absence of FH causes the C57BL6 mouse to become susceptible to chronic serum sickness (CSS) along with an increase in the renal infiltration of macrophages compared to controls. To understand if the increased recruitment of macrophages (Mϕs) to the kidney was driving inflammation and propagating injury, we examined the effect of Mϕ depletion with clodronate in FH knockout mice with CSS. Eight-week-old FHKO mice were treated with apoferritin (4 mg/mouse) for 5 wks and with either vehicle (PBS) or clodronate (50 mg/kg ip, 3 times/wk for the last 3 weeks). The administration of clodronate decreased monocytes and Mϕs in the kidneys by >80%. Kidney function assessed by BUN and albumin remained closer to normal on depletion of Mϕs. Clodronate treatment prevented the alteration in cytokines, TNFα and IL-6, and increase in gene expression of connective tissue growth factor (CTGF), TGFß-1, matrix metalloproteinase-9 (MMP9), fibronectin, laminin, and collagen in FHKO mice with CSS (P < 0.05). Clodronate treatment led to relative protection from immune complex- (IC-) mediated disease pathology during CSS as assessed by the significantly reduced glomerular pathology (GN) and extracellular matrix. Our results suggest that complement activation is one of the mechanism that regulates the macrophage landscape and thereby fibrosis. The exact mechanism remains to be deciphered. In brief, our data shows that Mϕs play a critical role in FH-dependent ICGN and Mϕ depletion reduces disease progression.

A promising dual-drug targeted delivery system in cancer therapy: nanocomplexes of folate-apoferritin-conjugated cationic solid lipid nanoparticles.

Various nano-sized protein and lipid complexes are being investigated as drug delivery systems. The encapsulation of more than one drug in a single nanocomplex carrier could enhance the therapeutic potency and afford synergistic therapeutic effects. In this study, we developed a novel protein-lipid nanocomplex as a controlled drug delivery system for two important cancer drugs, doxorubicin (DOX) and mitoxantrone (MTO). Apoferritin (AFr) functionalized with folic acid (FA) was used to encapsulate DOX to create the targeted protein nanocomplexes (TPNs). The second drug, MTO, was loaded into the cationic solid lipid nanoparticles (cSLN) to form the liposomal drug nanocomplex particles (MTO-cSLNs). Two complexes were then assembled by tight coupling through ionic interactions to obtain the final drug delivery system, the dual-targeted protein-lipid nanocomplexes (DTPLNs). UV-Vis and fluorescence spectroscopy were used for structural characterization of TPNs and DTPLNs. Transmission electron microscopy (TEM) was used for comprehensive analysis of the final DTPLNs. We confirmed that the DTPLNs display desired time-dependent and pH-dependent drug release behaviors. We also demonstrated the improved anti-cancer efficacy of DOX and MTO in their encapsulated DTPLNs as compared to their free forms. Our results provide promising prospects for the application of the DTPLNs as efficient drug delivery systems.

Developing a Novel Gold(III) Agent to Treat Glioma Based on the Unique Properties of Apoferritin Nanoparticles: Inducing Lethal Autophagy and Apoptosis.

Effective delivery of anticancer agents across the blood-brain barrier (BBB) required innovative strategies to achieve glioma regression. To resolve this problem, we proposed to develop a metal agent that target and treat glioma based on the unique property of apoferritin (AFt) nanoparticles (NPs). Thus, we synthesized a series of Au(III) 3-(4-metyl piperidine)thiosemicarbazides compounds and analyzed their structure-activity relationships, obtaining a Au agent (C6) with remarkable cytotoxicity in glioma. Moreover, we confirmed that C6 kills glioma cells by inducing lethal autophagy and apoptosis. Importantly, our results revealed that the successfully constructed apoferritin-C6 NPs (AFt-C6 NPs) can effectively cross the BBB, inhibit glioma growth, and selectively accumulate in tumors.

Tie2-mediated vascular remodeling by ferritin-based protein C nanoparticles confers antitumor and anti-metastatic activities.

BACKGROUND: Conventional therapeutic approaches for tumor angiogenesis, which are primarily focused on the inhibition of active angiogenesis to starve cancerous cells, target the vascular endothelial growth factor signaling pathway. This aggravates hypoxia within the tumor core and ultimately leads to increased tumor proliferation and metastasis. To overcome this limitation, we developed nanoparticles with antiseptic activity that target tumor vascular abnormalities. METHODS: Ferritin-based protein C nanoparticles (PCNs), known as TFG and TFMG, were generated and tested in Lewis lung carcinoma (LLC) allograft and MMTV-PyMT spontaneous breast cancer models. Immunohistochemical analysis was performed on tumor samples to evaluate the tumor vasculature. Western blot and permeability assays were used to explore the role and mechanism of the antitumor effects of PCNs in vivo. For knocking down proteins of interest, endothelial cells were transfected with siRNAs. Statistical analysis was performed using one-way ANOVA followed by post hoc Dunnett's multiple comparison test. RESULTS: PCNs significantly inhibited hypoxia and increased pericyte coverage, leading to the inhibition of tumor growth and metastasis, while increasing survival in LLC allograft and MMTV-PyMT spontaneous breast cancer models. The coadministration of cisplatin with PCNs induced a synergistic suppression of tumor growth by improving drug delivery as evidenced by increased blood prefusion and decreased vascular permeability. Moreover, PCNs altered the immune cell profiles within the tumor by increasing cytotoxic T cells and M1-like macrophages with antitumor activity. PCNs induced PAR-1/PAR-3 heterodimerization through EPCR occupation and PAR-1 activation, which resulted in Gα13-RhoA-mediated-Tie2 activation and stabilized vascular tight junctions via the Akt-FoxO3a signaling pathway. CONCLUSIONS: Cancer treatment targeting the tumor vasculature by inducing antitumor immune responses and enhancing the delivery of a chemotherapeutic agent with PCNs resulted in tumor regression and may provide an effective therapeutic strategy.

Co-administration of H-ferritin-doxorubicin and Trastuzumab in neoadjuvant setting improves efficacy and prevents cardiotoxicity in HER2 + murine breast cancer model.

Neoadjuvant chemotherapy has been established as the standard of care for HER2-positive breast cancer since it allows cancer down-staging, up to pathological complete response. The standard of care in the neoadjuvant setting for HER2-positive breast cancer is a combination of highly cytotoxic drugs such as anthracyclines and the anti-HER2 monoclonal antibody. Despite this cocktail allows a pathological complete response in up to 50%, their co-administration is strongly limited by intrinsic cardiotoxicity. Therefore, only a sequential administration of anthracyclines and the anti-HER2 treatment is allowed. Here, we propose the anthracycline formulation in H-Ferritin nanocages as promising candidate to solve this unmet clinical need, thanks to its capability to increase anthracyclines efficacy while reducing their cardiotoxicity. Treating a murine model of HER2-positive breast cancer with co-administration of Trastuzumab and H-Ferritin anthracycline nanoformulation, we demonstrate an improved tumor penetration of drugs, leading to increased anticancer efficacy and reduced of cardiotoxicity.

Peritumoral administration of IFNß upregulated mesenchymal stem cells inhibits tumor growth in an orthotopic, immunocompetent rat glioma model.

BACKGROUND: Immunotherapy with IFNß is a promising strategy for treating malignant glioma. However, systemic administration of IFNß is inadequate because of low intratumoral concentration and major adverse effects. This study aimed to determine whether mesenchymal stem cells (MSCs) can be used as cellular vehicles to locally deliver IFNß for glioma therapy by using in vivo MRI tracking. METHODS: A recombinant lentiviral vector encoding IFNß and ferritin heavy chain (FTH) reporter genes was constructed to transduce MSCs. The effectiveness and safety of transduction were assessed. After the IFNß and FTH overexpressed MSCs (IFNß-FTH-MSCs) were transplanted into intracranial orthotopic rat F98 gliomas via peritumoral, intracerebral, intratumoral or intra-arterial injection, MRI was performed to track IFNß-FTH-MSCs and to evaluate their therapeutic effect on glioma in vivo, as validated by histologic analysis, quantitative PCR and ELISA assays. RESULTS: MSCs were efficiently and safely transduced to upregulate their IFNß secretion and FTH expression by the constructed lentivirus. After peritumoral injection, IFNß-FTH-MSCs appeared as hypointense signals on MRI, which gradually diminished but remained visible until 11 days. Compared with other administration routes, only peritumoral injection of IFNß-FTH-MSCs showed a remarkable inhibition on the glioma growth. Nearly 30% of IFNß-FTH-MSCs survived up to 11 days after peritumoral injection, while most of IFNß-FTH-MSCs injected via other routes died within 11 days. IFNß-FTH-MSCs grafted peritumorally secreted IFNß persistently, leading to pronounced Batf3+ dendritic cells and CD8+ T lymphocyte infiltration within the glioma. CONCLUSIONS: MSCs can be used as cellular vehicles of IFNß to treat malignant glioma effectively via peritumoral injection.

Efficient synergistic combination effect of Quercetin with Curcumin on breast cancer cell apoptosis through their loading into Apo ferritin cavity.

Combination of natural agents has received a great attention in cancer treatment because of synergistically increased apoptotic effect on cancer cell lines by triggering several apoptotic signaling pathways. However, the hydrophobic nature, poor bioavailability and low cellular uptake of most natural agents limit their therapeutic effectiveness. The purpose of this study was to design Apoferritin nanoparticles loaded with Quercetin and Curcumin (Que-Cur-HoS-Apo NPs) and to test their synergistic antitumor properties on a breast cancer cell line (MCF7). The physico-chemical characterization of the Que-Cur-HoS-Apo NPs by Size Exclusion Chromatography (FPLC) and Dynamic Light Scattering (DLS) confirmed the encapsulation of the compounds in the protein cage with narrow size distribution in the range 17.4 ±â€¯1.2 nm. Cell viability study indicated that Que-Cur-HoS-Apo NPs were able to exert a more pronounced effect at lower dose on the MCF7 cell line when compared to the free combination of the drugs. The Que-Cur-HoS-Apo system allowed cellular uptake of natural agents thus triggering enhanced apoptosis. These effects were confirmed by Annexin-V/7-AAD Staining Assay and intracellular Reactive Oxygen Species (ROS) quantitative detection. These results suggest the potential of Que-Cur-HoS-Apo NPs as a promising anti-cancer agent in breast cancer therapy and pave the way to examine Que-Cur-HoS-Apo NPs effect in vivo.

Enhanced Copper-Temozolomide Interactions by Protein for Chemotherapy against Glioblastoma Multiforme.

Current treatment of recurrent glioblastoma multiforme (GBM) demands dose-intense temozolomide (TMZ), a prodrug of 5-(3-methyltriazen-1-yl) imidazole-4-carboxamide (MTIC), based on the spontaneous hydrolysis of TMZ at basic pH. However, how to control the activity of MTIC remains unknown, which poses a particular challenge to search a reliable MTIC receptor. We reported that copper, for the first time, is found to recognize and bind MTIC in the process of TMZ degradation, which means copper can play an important role in enhancing the bioavailability of MTIC derived from TMZ. Using apoferritin as a model copper-bound protein, we studied the copper-TMZ interaction in protein and observed efficient MTIC immobilization with high binding efficiency (up to 92.9% based on original TMZ) and capacity (up to 185 MTIC moieties per protein). The system was stable against both alkaline and acidic pH and could be activated by glutathione to liberate MTIC, which paves a way to deliver a DNA-alkylating agent for both TMZ-sensitive and TMZ-resistant GBM chemotherapy. Our study provides a new insight for understanding the potential relationship between the special GBM microenvironment (specific copper accumulation) and the therapeutic effect of TMZ.

GRP78-targeted ferritin nanocaged ultra-high dose of doxorubicin for hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer deaths, primarily due to its high incidence of recurrence and metastasis. Considerable efforts have therefore been undertaken to develop effective therapies; however, effective anti-HCC therapies rely on identification of suitable biomarkers, few of which are currently available for drug targeting. Methods: GRP78 was identified as the membrane receptor of HCC-targeted peptide SP94 by immunoprecipitation and mass spectrum analysis. To develop an effective anti-HCC drug nanocarrier, we first displayed GRP78-targeted peptide SP94 onto the exterior surface of Pyrococcus furiosus ferritin Fn (HccFn) by genetic engineering approach, and then loaded doxorubicin (Dox) into the cavities of HccFn via urea-based disassembly/reassembly method, thereby constructing a drug nanocarrier called HccFn-Dox. Results: We demonstrated that HccFn nanocage encapsulated ultra-high dose of Dox (up to 400 molecules Dox/protein nanocage). In vivo animal experiments showed that Dox encapsulated in HccFn-Dox was selectively delivered into HCC tumor cells, and effectively killed subcutaneous and lung metastatic HCC tumors. In addition, HccFn-Dox significantly reduced drug exposure to healthy organs and improved the maximum tolerated dose by six-fold compared with free Dox. Conclusion: In conclusion, our findings clearly demonstrate that GRP78 is an effective biomarker for HCC therapy, and GRP78-targeted HccFn nanocage is effective in delivering anti-HCC drug without damage to healthy tissue.

Endoscopic molecular imaging of early gastric cancer using fluorescently labeled human H-ferritin nanoparticle.

Optical imaging technologies improve clinical diagnostic accuracy of early gastric cancer (EGC). However, there was a lack of imaging agents exhibiting molecular specificity for EGCs. Here, we employed the dye labeled human heavy-chain ferritin (HFn) as imaging nanoprobe, which recognizes tumor biomarker transferrin receptor 1 (TfR1), to enable specific EGC imaging using confocal laser endomicroscopy (CLE). TfR1 expression was initially examined in vitro in gastric tumor cells and in vivo through whole-body fluorescence and CLE imaging in tumor-bearing mice. Subsequently, dye labeled HFn was topically applied to resected human tissues for EGC detection. CLE analysis of TfR1-targeted fluorescence imaging allowed distinction of neoplastic from non-neoplastic tissues (P < 0.0001), and TfR1 expression level was found to correlate with EGC differentiation degrees (P < 0.0001). Notably, the CLE evaluation correlated well with the immunohistochemical findings (κ-coefficient: 0.8023). Our HFn-nanoprobe-based CLE increases the accuracy of EGC detection and enables visualization of tumor margins and endoscopic resection.

Near-Infrared Fluorescent Dye-Decorated Nanocages to Form Grenade-like Nanoparticles with Dual Control Release for Photothermal Theranostics and Chemotherapy.

Recently, nanoparticles (NPs) have been widely investigated for delivery of anticancer drugs. Here, a dual control drug-release modality was developed that uses naturally occurring protein apoferritin loaded with doxorubicin (DOX) and ADS-780 near-infrared (NIR) fluorescent dye-decorated NPs (ADNIR NPs). ADNIR NPs act as a grenade to detonate the targeted tumor site following laser irradiation (photothermal therapy, PTT) and explode into cluster warheads (apoferritin-loaded DOX nanocages, AF-DOX NCs) that further destroy the tumor cells (chemotherapy). Light was shown to disrupt the grenade-like structure of NPs to release AF-DOX NCs as well as DOX from NCs in low-pH intercellular environments. In vitro and in vivo studies showed that the structure of AF-DOX NCs was disassembled to release DOX, which then killed the cancer cells in organelles with acidic environments. In vivo studies showed that the ADNIR NP-decorated with NIR dye facilitated tracking of the accumulated NPs at the tumor site using an IVIS imaging system. Overall, targeted ADNIR NPs with dual-release mechanisms were developed for use in photothermal theranostic and chemotherapy. This modality has high potential for application in cancer treatment and clinical translation for drug delivery and imaging.

The presence of glutamate residues on the PAS sequence of the stimuli-sensitive nano-ferritin improves in vivo biodistribution and mitoxantrone encapsulation homogeneity.

A genetically engineered human ferritin heavy chain (HFt)-based construct has been recently shown by our group to efficiently entrap and deliver doxorubicin to cancer cells. This construct, named HFt-MP-PAS, contained a tumor-selective sequence (MP) responsive to proteolytic cleavage by tumor proteases (MMPs), located between each HFt subunit and an outer shielding polypeptide sequence rich in proline (P), serine (S) and alanine (A) residues (PAS). HFt-MP-PAS displayed excellent therapeutic efficacy in xenogenic pancreatic and head and neck cancer models in vivo, leading to a significant increase in overall animal survivals. Here we report a new construct obtained by the genetic insertion of two glutamate residues in the PAS sequence of HFt-MP-PAS. Such new construct, named HFt-MP-PASE, is characterized by improved performances as drug biodistribution in a xenogenic pancreatic cancer model in vivo. Moreover, HFt-MP-PASE efficiently encapsulates the anti-cancer drug mitoxantrone (MIT), and the resulting MIT-loaded nanoparticles proved to be more soluble and monodispersed than the HFt-MP-PAS counterparts. Importantly, in vitro MIT-loaded HFt-MP-PASE kills several cancer cell lines of different origin (colon, breast, sarcoma and pancreas) at least as efficiently as the free drug. Finally, our MIT loaded protein nanocages allowed in vivo an impressive incrementing of the drug accumulation in the tumor with respect to the free drug.

Apoferritin as an ubiquitous nanocarrier with excellent shelf life.

Due to many adverse effects of conventional chemotherapy, novel methods of targeting drugs to cancer cells are being investigated. Nanosize carriers are a suitable platform for this specific delivery. Herein, we evaluated the long-term stability of the naturally found protein nanocarrier apoferritin (Apo) with encapsulated doxorubicin (Dox). The encapsulation was performed using Apo's ability to disassemble reversibly into its subunits at low pH (2.7) and reassemble in neutral pH (7.2), physically entrapping drug molecules in its cavity (creating ApoDox). In this study, ApoDox was prepared in water and phosphate-buffered saline and stored for 12 weeks in various conditions (-20°C, 4°C, 20°C, and 37°C in dark, and 4°C and 20°C under ambient light). During storage, a very low amount of prematurely released drug molecules were detected (maximum of 7.5% for ApoDox prepared in PBS and 4.4% for ApoDox prepared in water). Fourier-transform infrared spectra revealed no significant differences in any of the samples after storage. Most of the ApoDox prepared in phosphate-buffered saline and ApoDox prepared in water and stored at -20°C formed very large aggregates (up to 487% of original size). Only ApoDox prepared in water and stored at 4°C showed no significant increase in size or shape. Although this storage caused slower internalization to LNCaP prostate cancer cells, ApoDox (2.5 µM of Dox) still retained its ability to inhibit completely the growth of 1.5×104 LNCaP cells after 72 hours. ApoDox stored at 20°C and 37°C in water was not able to deliver Dox inside the nucleus, and thus did not inhibit the growth of the LNCaP cells. Overall, our study demonstrates that ApoDox has very good stability over the course of 12 weeks when stored properly (at 4°C), and is thus suitable for use as a nanocarrier in the specific delivery of anticancer drugs to patients.

Selective delivery of doxorubicin by novel stimuli-sensitive nano-ferritins overcomes tumor refractoriness.

Human ferritin heavy chain (HFt) has been demonstrated to possess considerable potential for targeted delivery of drugs and diagnostic agents to cancer cells. Here, we report the development of a novel HFt-based genetic construct (HFt-MP-PAS) containing a short peptide linker (MP) between each HFt subunit and an outer shielding polypeptide sequence rich in proline (P), serine (S) and alanine (A) residues (PAS). The peptide linker contains a matrix-metalloproteinases (MMPs) cleavage site that permits the protective PAS shield to be removed by tumor-driven proteolytic cleavage within the tumor microenvironment. For the first time HFt-MP-PAS ability to deliver doxorubicin to cancer cells, subcellular localization, and therapeutic efficacy on a xenogeneic mouse model of a highly refractory to conventional chemotherapeutics type of cancer were evaluated. HFt-MP-PAS-DOXO performance was compared with the novel albumin-based drug delivery system INNO-206, currently in phase III clinical trials. The results of this work provide solid evidence indicating that the stimuli-sensitive, long-circulating HFt-MP-PAS nanocarriers described herein have the potential to be exploited in cancer therapy.

Different doxorubicin formulations affect plasma 4-hydroxy-2-nonenal and gene expression of aldehyde dehydrogenase 3A1 and thioredoxin reductase 2 in rat.

Increased oxidative stress is indisputably an important mechanism of doxorubicin side effects, especially its cardiotoxicity. To prevent impairment of non-tumorous tissue and to improve the specificity in targeting the tumor tissue, new drug nanotransporters are developed. In many cases preclinical therapeutic advantage has been shown when compared with the administration of conventional drug solution. Three forms of doxorubicin--conventional (DOX), encapsulated in liposomes (lipoDOX) and in apoferritin (apoDOX) were applied to Wistar rats. After 24 h exposition, the plasma level of 4-hydroxy-2-nonenal (4-HNE) as a marker of lipoperoxidation and tissue gene expression of thioredoxin reductase 2 (TXNRD2) and aldehyde dehydrogenase 3A1 (ALDH3A1) as an important part of antioxidative system were determined. Only conventional DOX significantly increases the level of 4-HNE; encapsulated forms on the other hand show significant decrease in plasma levels of 4-HNE in comparison with DOX. They also cause significant decrease in gene expression of ALDH3A1 and TXNRD2 in liver as a main detoxification organ, and a mild influence on the expression of these enzymes in left heart ventricle as a potential target of toxicity. Thus, 4-HNE seems to be a good potential biomarker of oxidative stress induced by various forms of doxorubicin.

Hyaluronic acid-conjugated apoferritin nanocages for lung cancer targeted drug delivery.

In this paper, we proposed a naturally derived protein cage based pH-responsive delivery system for intracellular prodrug controlled release. The drug delivery system is based on apoferritin as delivery vehicles to encapsulate the anticancer drug daunomycin (DN) and alleviate the side effect. The hydrophobic drug DN was encapsulated into the interior of apoferritin by the hydrophobic channels of the cage with swelling at slight acidic pH and electrostatic adsorption. The negatively charged poly-l-aspartic acid (PLAA) was further introduced into the apoferritin to absorb the positively charged DN. The mixture of PLAA and DN easily flew into the apoferritin cage and was stably stored in the physiological fluids. PLAA protected the leakage of DN and encapsulated a sufficient amount of drug molecules in the cage. To specifically target the tumor cells, the surface of apoferritin was modified with hyaluronic acid (HA) which can easily bind to the HA-receptor CD44. Here, human embryonic lung MRC-5 cells and lung cancer A549 cells were used to observe the specific binding of HA and morphological changes in vitro and examine the antitumor activity. This unique protein based drug delivery platform using the apoferritin cage shows great potential in the therapeutic administration of the anti-cancer agents.

Protein nanocages for self-triggered nuclear delivery of DNA-targeted chemotherapeutics in Cancer Cells.

A genetically engineered apoferritin variant consisting of 24 heavy-chain subunits (HFn) was produced to achieve a cumulative delivery of an antitumor drug, which exerts its cytotoxic action by targeting the DNA at the nucleus of human cancer cells with subcellular precision. The rationale of our approach is based on exploiting the natural arsenal of defense of cancer cells to stimulate them to recruit large amounts of HFn nanoparticles loaded with doxorubicin inside their nucleus in response to a DNA damage, which leads to a programmed cell death. After demonstrating the selectivity of HFn for representative cancer cells compared to healthy fibroblasts, doxorubicin-loaded HFn was used to treat the cancer cells. The results from confocal microscopy and DNA damage assays proved that loading of doxorubicin in HFn nanoparticles increased the nuclear delivery of the drug, thus enhancing doxorubicin efficacy. Doxorubicin-loaded HFn acts as a "Trojan Horse": HFn was internalized in cancer cells faster and more efficiently compared to free doxorubicin, then promptly translocated into the nucleus following the DNA damage caused by the partial release in the cytoplasm of encapsulated doxorubicin. This self-triggered translocation mechanism allowed the drug to be directly released in the nuclear compartment, where it exerted its toxic action. This approach was reliable and straightforward providing an antiproliferative effect with high reproducibility. The particular self-assembling nature of HFn nanocage makes it a versatile and tunable nanovector for a broad range of molecules suitable both for detection and treatment of cancer cells.

Curcumin/Gd loaded apoferritin: a novel "theranostic" agent to prevent hepatocellular damage in toxic induced acute hepatitis.

Apoferritin has been exploited to deliver simultaneously therapeutic and imaging agents (loaded into its internal cavity) to hepatocytes as this protein is efficiently taken up from blood by hepatocyte scavenger receptor class A type 5 via the ferritin transporting route. To this purpose the protein has been loaded with the magnetic resonance imaging (MRI) contrast agent GdHPDO3A and curcumin, a polyphenolic substance endowed with multiple pharmacological actions, namely: antioxidant, anti-inflammatory, antineoplastic. Curcumin and GdHPDO3A loaded apoferritin has been used with the aim to attenuate the thioacetamide-induced hepatitis together with the evaluation by MRI of drug delivery efficiency. Mice pretreated by intraperitoneal administration showed significantly attenuated hepatic injury as assessed by measuring alanine aminotransferase (ALT) activity in plasma and by histology assessment. The encapsulation of curcumin inside the apoferritin cavity significantly increases its stability and bioavailability while maintaining its therapeutic anti-inflammatory properties.

Curcumin alleviates immune-complex-mediated glomerulonephritis in factor-H-deficient mice.

Complement factor H (Cfh) is a key regulator of the complement cascade and protects C57BL/6 mice from immune complex-mediated complement-dependent glomerulonephritis. In chronic serum sickness (CSS) there are increased deposits of immune complexes in the glomeruli with inflammation and a scarring phenotype. As cucurmin is an effective anti-inflammatory agent and reduces complement activation, we hypothesized that it should alleviate renal disease in this setting. To determine the effectiveness of curcumin, an apoferritin-induced CSS model in Cfh-deficient (Cfh(-/-)) mice was used. Curcumin treatment (30 mg/kg) given every day in parallel with apoferritin reduced glomerulonephritis and enhanced kidney function (blood urea nitrogen, 45·4 ± 7·5 versus 35·6 ± 5·1; albuminuria, 50·1 ± 7·1 versus 15·7 ± 7·1; glomerulonephritis, 2·62 + 0·25 versus 2 + 0·3, P < 0·05). In line with reduced IgG deposits in mice with CSS given curcumin, C9 deposits were reduced indicating reduced complement activation. Mice treated with curcumin had a significant reduction in the number of splenic CD19(+) B cells and the ratio of CD19 : CD3 cells (P < 0·05) with no change in the T-cell population. Myeloperoxidase assay showed reduced macrophages in the kidney. However, a significant reduction in the M2 subset of splenic macrophages by apoferritin was prevented by curcumin, suggesting a protective function. Curcumin treatment reduced mRNA expression of inflammatory proteins monocyte chemoattractant protein-1 and transforming growth factor-ß and matrix proteins, fibronectin, laminin and collagen. Our results clearly illustrate that curcumin reduces glomerulosclerosis, improves kidney function and could serve as a therapeutic agent during serum sickness.

Characterization and cellular uptake of platinum anticancer drugs encapsulated in apoferritin.

Clinical application of platinum-based anticancer drugs is largely limited by severe general toxicity and drug resistance. Drug delivery systems with tumor-targeting potential are highly desired for improving the efficacy and applicability of these drugs. This study describes an alternative strategy for the delivery of platinum drugs (cisplatin, carboplatin and oxaliplatin) by encapsulating each of them in the cavity of apoferritin (AFt). The encapsulation was achieved through manipulating the pH-dependent unfolding-refolding process of AFt at pH 2.0 and 7.4, respectively, in saturated drug solution. UV-vis spectrometry, circular dichroism spectrometry, dynamic light scattering, and inductively coupled plasma mass spectrometry were used to characterize the AFt-drug complexes. The loading capacity of AFt varies with respective drugs and the structural integrity of the protein shell remains intact after encapsulation. In vitro assays on the rat pheochromocytoma cell line (PC12) show that AFt-cisplatin inhibits the cells in a slow but sustaining mode and the cellular uptake of platinum is enhanced by AFt. AFt-carboplatin and AFt-oxaliplatin complexes only exhibit a marginal cytotoxicity towards this cell line under similar concentrations.