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. 2013 Jan 8;21(3):533–541. doi: 10.1038/mt.2012.256

Therapeutic Assessment of Cytochrome C for the Prevention of Obesity Through Endothelial Cell-targeted Nanoparticulate System

Md Nazir Hossen 1, Kazuaki Kajimoto 1, Hidetaka Akita 2, Mamoru Hyodo 1, Taichi Ishitsuka 2, Hideyoshi Harashima 1,2,*
PMCID: PMC3589155  PMID: 23295953

Abstract

Because the functional apoptosis-initiating protein, cytochrome C (CytC) is rapidly cleared from the circulation (t1/2 (half-life): 4 minutes), it cannot be used for in vivo therapy. We report herein on a hitherto unreported strategy for delivering exogenous CytC as a potential and safe antiobesity drug for preventing diet-induced obesity, the most common type of obesity in humans. The functional activity of CytC encapsulated in prohibitin (a white fat vessel-specific receptor)-targeted nanoparticles (PTNP) was evaluated quantitatively, as evidenced by the observations that CytC-loaded PTNP causes apoptosis in primary adipose endothelial cells in a dose-dependent manner, whereas CytC alone did not. The delivery of a single dose of CytC through PTNP into the circulation disrupted the vascular structure by the targeted apoptosis of adipose endothelial cells in vivo. Intravenous treatment of CytC-loaded PTNP resulted in a substantial reduction in obesity in high-fat diet (HFD) fed wild-type (wt) mice, as evidenced by the dose-dependent prevention of the percentage of increase in body weight and decrease in serum leptin levels. In addition, no detectable hepatotoxicity was found to be associated with this prevention. Thus, the finding highlights the promising potential of CytC for use as an antiobesity drug, when delivered through a nanosystem.

Introduction

Obesity, the deposition of excess fat in the body, is recognized as a growing medical problem worldwide.1 The current pharmacotherapeutic management of obesity is limited to one drug: Orlistat. This drug, however, is also associated with low efficacy (≤5%) and has numerous side effects, including severe diarrhea, acute kidney toxicity, and malabsorption of vital vitamins.1,2,3 Therefore, it is likely that it will not be possible to manage this growing epidemic disease using this drug, and there is great urgency and need for introducing a novel, safe, and effective antiobesity drug.

White fat (WF) exhibits angiogenic activity.4,5 It was reported that the expansion and growth of WF is highly correlated with the formation of new adipose vessel.4,5,6,7 Consequently, antiangiogenic drugs such as TNP-470, matrix metalloproteinase inhibitors, polyphenol compounds, proapoptotic peptide (D(KLAKLAK)2, KLA), etc. have shown promise for weight reduction and adipose tissue loss in various models of obesity.8,9,10,11,12 Their results clearly established that ablation or inhibition of neovascularization via apoptosis/necrosis of endothelial cells in WF has the potential to inhibit the formation of new adipocytes, adipocyte tissue hypertrophy and ultimately, the development of obesity. However, owing to the generally pleiotropic activity of conventional chemotherapeutics, the use of these drugs is associated with neurotoxicity, nephrotoxicity, and cardiotoxicity, even though they are administered at their respective therapeutic dosage.9,13,14 Therefore, the selective targeting of adipose endothelial cells by pharmacological manipulation through nanoparticulate-targeted system offers an attractive therapeutic avenue for the effective management of obesity.

Cytochrome C (CytC), either endogenous release or exogenous delivery into cell cytoplasm executes its apoptotic role through activation of a caspase cascade.15,16,17 Thus, the use of this biomacromolecule may be advantageous for the development of a potential novel therapeutic system; (i) CytC, itself triggers the release of it through stimulating the release of calcium from the endoplasmic reticulum and costimulates its apoptotic activity, (ii) it is nontoxic because of its impermeability to the cell membrane, and is readily cleared from the circulation (t1/2 (half-life): 4 minutes).15,16,17,18,19 However, to allow its eventual interaction with corresponding targeted moieties in the cytoplasm of cells, exogenous CytC must be shielded from external inactivating effects in vivo and subsequently be delivered to its site of action.

In an attempt to develop targeted therapy for the control of obesity, Kolonin, MG et al. previously originally reported on prohibitin as a vascular receptor for the circular peptide motif (CKGGRAKDC) in WF of wild-type (wt) and lepob/ob mice.12 Regarding the adoption of an alternative adipose vascular-targeting strategy, we recently reported the preparation of an adipose endothelial cell-targeted nanoparticle in vitro.20,21 For in vivo applications, it was further optimized by the incorporation of three elements on its surface and this was regarded as prohibitin-targeted nanoparticle (PTNP). The PTNP system was designed in such a way so that (i) a PEGylated lipid bilayer provided the protection of encapsulated therapeutic cargos against biodegradation, (ii) a prohibitin-homing peptide ligand specifically and potentially delivered its payload to the cytosol of endothelial cells in WF vessel (WFV), and (iii) a long polyethylene glycol (PEG)-spacer between ligand and the surface of PEGylated nanoparticle assisted to reduce steric hindrance for multivalent active ligand-receptor recognition.22

Considering that the PTNP system would be able to specifically and potentially deliver a therapeutic cargo to the endothelial cells of WFV, we propose herein a highly biodegradable protein, CytC as an effective and safe antiobesity drug. Interestingly, CytC-induced apoptosis of endothelial cells of WFV in vitro and in vivo when it was delivered through the PTNP system and the CytC-loaded PTNP prevented diet-induced obesity (DIO) in a dose-dependent manner without any detectable liver toxicity. It can therefore be concluded that CytC is a potentially promising drug for the effective and safe prevention of DIO, the most common type obesity in humans.

Results

Physicochemical characterization of empty- and CytC-loaded PTNP

The average nanoparticle sizes and polydispersity index of empty- and CytC-loaded PTNP were similar (around 100 nm, polydispersity index <0.3) (Table 1). Nanoparticles with average diameter of around 100 nm may be suitable for the extended retention of them into the blood circulation for delivering the drug to its targeted cells.23 The nanoparticles without or with CytC had shown almost neutral (0.3 ± 2.5 and 2.5 ± 5.9 mV) zeta potentials, respectively (Table 1). The similar zeta potentials of empty- and CytC-loaded PTNP suggested that CytC retained maximally within the PTNP system as an aqueous phase since CytC with an isoelectric point (pI 10.5) and with an affinity to associate with neutral lipids would have facilitated its encapsulation into nanoparticles without electrostatic interaction with lipid membrane.24,25

Table 1. Physicochemical properties of empty- and CytC-loaded PTNP.

graphic file with name mt2012256t1.jpg

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Evaluation of the functional activity of CytC-loaded PTNP on primary endothelial cells derived from murine adipose tissue in vitro

In order to evaluate the functional activity of CytC via the cytoplasmic delivery of its active form by PTNP into primary endothelial cells isolated from murine adipose tissue (pcEC-IWAT), we performed a series of quantitative and qualitative in vitro apoptosis experiments. Treatment with various doses of CytC-loaded PTNP (3, 6, and 12 µg/ml of CytC) induced the activation of caspase 9, indicating the apoptosis of pcEC-IWAT cells in a dose-dependent manner, whereas the controls (free CytC at same doses and placebo-treated cells) did not (Figure 1a). In addition, apoptotic cells that were characterized by round shape, cell shrinkage, membrane blebbing, nucleus DNA fragmentation, and condensation of pcEC-IWAT cells, were observed when CytC-loaded PTNP was used at a dose of 6 µg/ml of CytC, whereas the controls did not (Figure 1b). These data indicate that CytC retained its active form into cytosol of pcEC-IWAT cells and that it caused apoptosis of these cells in a dose-dependent manner, only when CytC was delivered through the PTNP system.

Figure 1.

Figure 1

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Evaluation of functional activity of CytC-loaded PTNP on primary endothelial cells derived from murine adipose tissue in vitro. (a) pEC-IWAT cells (5 × 104 cells/well) were treated with CytC-loaded PTNP (3, 6, and 12 µg/ml of CytC) and controls (empty PTNP and free CytC at the same doses) for 3 hours to induce apoptosis. A mixture of equal volumes of caspase-Glo 9 reagent and cell lysate was incubated for ~1 hour and the activity of caspase 9 was measured based on luminescence (RLU) (n = 4). **P < 0.01 (CytC-loaded PTNP compared with free CytC) (Student's t-test). (b) pcEC-IWAT cells (2 × 105 cells/well) were treated with CytC-loaded PTNP (6 µg/ml of CytC) and controls (nontreated, empty PTNP, and free CytC at the same dose) for 6 hours for the induction of apoptosis. Nuclei (blue) were also stained with Hoechst 33342 and observed by CLSM. Bar = 20 µm. CLSM, confocal laser scanning microscope; CytC, cytochrome C; DIC, differential interference contrast; NS, not significant; PTNP, prohibitin-targeted nanoparticle; RLU, relative luminescence unit.

Moreover, we evaluated the cell-type specificity by the treatment of either the CytC-loaded PTNP system (6 µg/ml of CytC) or a physical mixture (empty PTNP (0.65 µmol) + CytC (6 µg/ml)) on pcEC-IWAT and NIH3T3 to induce apoptosis for 3 hours. The CytC-loaded PTNP system significantly induced caspase 9 activation in pcEC-IWAT cells, compared with the non-treatment control whereas, in the case of NIH3T3 cell, it did not. In addition, when both cells were treated with a physical mixture, no induction of caspase 9 activation was observed (Supplementary Figure S1).

Vascular disruption of adipose tissue with a single dose of CytC-loaded PTNP in vivo

In order to confirm the successful delivery of intact PTNP to WFV, double-labeled PTNP was intravenously injected into mice. After 6 hours, double-labeled PTNP was highly colocalized with vessels, as evidenced by the integration of NBD-DOPE (lipid membrane) and rhodamine (aqueous phase) in the same nanoparticle and with alexa647-labeled Griffonia simplicifolia isolectin B4 (GS-IB4) (vessels), while it was not detected in the vessels of other organs (lungs, heart, kidneys, and liver) (Figure 2a). Meanwhile, confocal observations of plasma fractions in blood indicated that the double-labeled PTNP remained in an intact state (colocalization of lipid membrane: NBD-DOPE and aqueous phase: rhodamine), as identified by yellow dots, which is analogous to long, circulating double-labeled PEGylated nanoparticles (double-labeled non-targeted nanoparticles (NTNP)) (Supplementary Figure S2a). In addition, the spectra of NBD-DOPE and rhodamine appeared in the same position and the intensity was quite similar, like those of double-labeled NTNP (Supplementary Figure S2b). Considering these results, the colocalization of the double-labeled PTNP to WFV indicates that PTNP remains intact in the circulation and therefore shows promise for use in delivering an aqueous phase marker to a target. Thus, mice were given a single intravenous injection of a dose of 6 mg/kg of CytC in lieu of aqueous phase marker-loaded PTNP. At 24 hours post-injection, WFV stained with GS-IB4 was colocalized with the green fluorescence (fluorescein isothiocyanate), indicating the existence of active caspase activity, whereas, in nontreated and empty PTNP-treated mice, only background fluorescence was observed (Figure 2b). In addition, the delivery of CytC through the PTNP system did not induce the apoptosis of endothelial cells in other organs such as heart, lung, liver, spleen, and kidney, whereas in the case of the physical mixture (empty PTNP + CytC) or nontreated, apoptosis was not found in the WFV (Supplementary Figure S3). These findings provide further evidence to show that the cytoplasmic delivery of active CytC via PTNP can cause adipose endothelial cell apoptosis in vivo.

Figure 2.

Figure 2

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Destruction of adipose vascular endothelial cell with a single dose of CytC-loaded PTNP in vivo. (a) Delivery of aqueous phase marker to adipose vessels. Double-labeled PTNP (lipid membrane: NBD-DOPE and aqueous phase: rhodamine) was intravenously injected with a total lipid dose of 0.1 mmol/kg body weight into mice. After 6 hours, tissue pieces of inguinal adipose tissue (IAT), lungs, heart, kidney, and liver were observed by confocal microscopy. Bar = 20 µm. (b) Vascular disruption occurred due to adipose endothelial cell apoptosis. Mice were untreated or received a single dose of empty or CytC-loaded PTNP (6 mg/kg) intravenously. We identified endothelial cells in the middle panel (red) and apoptotic cells in the left panel (green) in unfixed SAT. Cells stained with both markers appear yellow (right panel) indicate endothelial cell apoptosis. Bar = 100 µm. CytC, cytochrome C; FITC, fluorescein isothiocyanate; FLICA, fluorescent-labeled inhibitors of caspase; PTNP, prohibitin-targeted nanoparticle; SAT, subcutaneous adipose tissue.

Delivery of CytC-loaded PTNP decreases the progression of obesity burden

To determine whether the targeted delivery of CytC has any observable beneficial effects in preventing obesity, we then treated mice that had been exposed to a high-fat diet (HFD) at 3-day intervals for a total of 30 days with intravenous injections of 6, 1.2, and 0.25 mg/kg of the CytC-PTNP system or untreated (allowed to HFD and normal diet (ND)) as a pair of controls. The percentage of increase in body weight for the HFD-fed mice was significantly decreased when treated with the CytC-PTNP system respectively, compared with the HFD control group and the effect was dose-dependent (Figure 3). In addition, the treatment of mice with the CytC-loaded PTNP system did not decrease the cumulative energy intake, compared with the physical mixture and the nontreated (ND and HFD) mice (Supplementary Figure S4a), while the blood glucose concentration of all groups was comparable (Supplementary Figure S4b). These results provide strong evidence to show that treatment with CytC-loaded PTNP has the potential for use in preventing the development of obesity in mice that are fed HFD without any effect on food intake.

Figure 3.

Figure 3

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Delivery of CytC-loaded PTNP decreases the progression of obesity burden. Mice were exposed to a high-fat diet (HFD) along with an intravenous injection of 6, 1.2, and 0.25 mg/kg of CytC-loaded PTNP at 3-day intervals for 30 days respectively, whereas the other two groups were left untreated and allowed access to a HFD and a normal diet (ND) as a pair of controls. The body weights of each mouse were measured every 3 days before injections and the percentage of increase in body weight in treated mice as compared with untreated controls starting from the day of treatment (black arrow) are shown. Data are expressed as the mean ± SD. (n = 3); **P < 0.01, *P < 0.05 and NS, not significant versus HFD controls (Dunnett's multiple comparison test). CytC, cytochrome C; PTNP, prohibitin-targeted nanoparticle.

The prevention of DIO is associated with ablation of fat mass

To observe whether the targeted apoptosis of adipose endothelial cells by treatment with CytC-loaded PTNP is associated with the ablation of fat mass, we excised the subcutaneous adipose tissue (SAT) and epididymal adipose tissue (EAT) on day 30 of the treatment. The size and length of SAT and EAT showed a substantial decrease and resembled excised organs obtained from the ND-fed control group. In contrast, the size and length of these adipose tissues from the HFD control group were large (Figure 4a). Dissected SAT and EAT also showed that the mass of fat depots from treated mice were significantly reduced compared with the HFD control group (Figure 4b). These results indicated that targeted ablation of adipose endothelial cells via the CytC-PTNP system had the potential to reduce the increase in fat mass.

Figure 4.

Figure 4

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The prevention of diet-induced obesity is associated with the ablation of fat mass. On 30 days of the treatment, representative mice and adipose tissues were taken snaps and measured weights of excised subcutaneous (SAT) and epididymal (EAT) adipose tissues. (a) The appearance of representative treated (6 mg/kg/3 days), HFD and ND control mice and their fat depots excised from SAT and EAT regions. (b) Percentages of fat mass per body weight. Weights of EAT and SAT tissues were normalized by individual body weights. Data are expressed as the mean ± SD. (n = 3); **P < 0.01, *P < 0.05 and NS, not significant versus HFD controls (Student's t-test). HFD, high-fat diet; ND, normal diet.

Plasma parameters related to antiobesity

To further examine the effect of antiobesity related to biomolecules, we measured serum parameters. The HFD-fed control group showed high levels of leptin, whereas the levels for the ND-fed control group were maintained at normal levels (Figure 5a). Treatment with CytC-loaded PTNP suppressed the serum leptin levels in a dose-dependent manner (Figure 5a). The treated mice also showed a tendency to have retrofitted serum levels of tumor necrosis factor-α (TNF-α) compared with HFD controls, similar to the ND-fed control group (Figure 5b). The levels of triglycerides (TG), total cholesterol, and free fatty acids had a tendency to decrease in the treated mice compared with HFD-fed group (not statistically significant) (Figure 5c–e). These results suggested that CytC-loaded PTNP had an impact to retrofit the obesity-related parameters.

Figure 5.

Figure 5

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Plasma parameters related to antiobesity. After the treatment, serum samples from five groups were collected. (a) Serum leptin levels were determined using ELISA assays. (b) Tumor necrosis factor-α (TNF-α), (c) triglyceride, (d) total cholesterols (Chol), and (e) nonesterified free fatty acid (NEFA) in plasma of treated mice (6 mg/kg/3 days CytC-loaded PTNP) and ND controls were compared with the high-fat controls. Data are expressed as the mean ± SD (n = 3); **P < 0.01, *P < 0.05 compared with the high-fat diet controls (Dunnett's multiple comparison test). CytC, cytochrome C; HFD, high-fat diet; ND, normal diet; PTNP, prohibitin-targeted nanoparticle; TG, triglyceride.

Toxicological study

To investigate the toxicities associated with this therapy, we further measured the serum parameters that are involved in eliciting the toxicities of liver. The serum levels of liver enzyme, alanine aminotransferase (ALT), remained normal in mice that were treated with CytC-loaded PTNP (Figure 6).

Figure 6.

Figure 6

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Toxicological study. Serum alanine aminotransferase (ALT) in plasma of treated mice (6 mg/kg/3 days CytC-loaded PTNP), HFD and ND controls were compared with the high-fat controls. Data are expressed as the mean ± SD (n = 3); NS (nonsignificant), compared with the high-fat diet controls (Dunnett's multiple comparison test). CytC, cytochrome C; GPT, glutamic pyruvic transaminase; HFD, high-fat diet; ND, normal diet; PTNP, prohibitin-targeted nanoparticle.

Discussion

The current pharmacotherapeutic management for obesity is limited only to the use of Orlistat, a lipase inhibitor. In the present study, the therapeutic potential of CytC as a novel antiobesity drug through PTNP system for the prevention of DIO was investigated.

CytC is a physiologically adaptable mitochondrial protein. It plays an important role inmediating apoptosis via the exertion of multiple stimuli such as Bax, Ca2+ overload, oxidants, chemotherapeutics, and DNA-damaging agents on mitochondria. Upon such conditions, a permeability transition pore was formed in the outer mitochondrial membrane, thus releasing CytC from the intermembrane space of mitochondria into the cytosol. The liberated CytC initiates the formation of an apoptosome consisting of apoptotic protease-activating factor-1 oligomers. The apoptotic protease-activating factor-1 apoptosome recruits and activates caspase 9, which, in turn, activates the executioner caspases, caspase 3 and 7. The active executioners kill the cell by the proteolysis of key cellular substrates.26,27,28,29,30 In addition, since apoptosis is a physiological process in which apoptotic bodies are formed by degradation via the action of lytic enzymes, they can readily undergo clearance by lymphocytes and macrophages with minimal deleterious effects to the healthy surrounding tissue.26,27 Although an adequate level of cytoplasmic CytC can affirm apoptosis of that cell, the delivery of exogenous CytC to its active form in the cytoplasm of cell remains a great challenge in vivo.

In order to achieve the functional activity of this protein in obesity, it is necessary to deliver exogenous CytC into the cytoplasm of endothelial cells in adipose tissue after encapsulating it within nanoparticulate system, which can be targetable. PTNP system specifically accumulated into the vascular endothelial cells of adipose tissue in mice, while it was sparing the vessels of other organs such as brain, heart, lungs, kidneys, liver, and spleen.22 Therefore, it may be feasible that PTNP has the ability to specifically deliver its associated therapeutic cargo in the WFV in vivo. We next encapsulated CytC into nanoparticles and the resulting products were characterized (Table 1). Since CytC is a water soluble physiological protein with an isoelectric point (pI 10.5), it may be assumed that it would be readily encapsulated as an aqueous internal core into a nanoparticle with a diameter of around 100 nm. It was reported that hydrophilic proteins, including CytC, ovalbumin (pI 4.5), and horseradish peroxidase (pI 7.2) are likely to be physically loaded into nanoparticles without using electrostatic interactions between carrier and proteins.24 In addition, we found that the entrapped aqueous volume of CytC-PTNP estimated from the % recovery ratio (%RR) of lipid (33.9%) and CytC (15.23%) after ultracentrifugation was 64.5 µl/mg of lipids, suggesting that this high volume of entrapment of CytC/mg by lipids (egg yolk phosphatidylcholine and cholesterol) may be not only by the physical entrapment but also demonstrates the ability of CytC to interact with neutral lipids.24,25 However, the question of whether encapsulated CytC is an active or inactive state in nanocavities needs to be addressed. Therefore, we initially tested the feasibility of directly treating CytC with an organic solvent mixture (chloroform:diisopropyl ethanol, 1:1 (vol/vol)) which was utilized in the preparation of the PTNP system. The ratio of OD400 and OD280 for the treated CytC solution along with probe sonication at the same condition indicated that most of the CytC (~90%) remains in an active state without lipids in this process (Supplementary Figure S5). The active CytC-loaded PTNP was then applied on pcEC-IWAT for the quantitative evaluation of in vitro apoptosis, suggesting that cytoplasmic delivery of CytC through the PTNP system induced apoptosis of pcEC-IWAT in a dose-dependent manner (Figure 1a). Furthermore, the apoptosis of pcEC-IWAT cells by the treatment of CytC-loaded PTNP was confirmed by the altered endothelial cell characteristics, including round shape, DNA condensation, etc. that were consistent with the findings of apoptosis in other cell lines16,17 (Figure 1b). It is likely that PTNP binds to this endothelial cell through the receptor–ligand interaction, internalizes and delivers its content into cell cytoplasm to be escaped from endosomes/lysosomes.21 On the other hand, free CytC, due to its membrane impermeability and lack of appropriate machinery to enter into the cell, did not bind (Figure 1a,b). Moreover, in our preliminary examination, ovalbumin, a non-apoptosis–inducing protein, did not induce caspase 9 activation when it was delivered to pcEC-IWAT through the PTNP system (data not shown). These results demonstrate the functional ability of CytC to cause apoptosis of pcEC-IWAT in a dose-dependent manner.

CytC is a highly biodegradable protein with a biological half-life of 4 minutes. It is easily adsorbed by blood proteins and the subsequent interaction with phagocyte results in the rapid clearance from the body.19 Intactness of PTNP inside the circulation is one of the key features to deliver exogenous CytC as an aqueous internal core to the cytoplasm of vascular endothelial cells into adipose tissue since PTNP may offer a new identity of CytC by causing CytC take on the pharmacokinetic profiles of the carrier. Therefore, through double labeling of PTNP, we observed intact PTNP (an integrated lipid membrane and aqueous core in the same nanoparticle) on WFV (Figure 2a). As mentioned above, cytoplasmic delivery of CytC via PTNP to endothelial cells could activate caspase cascade even at very minute doses. We then tested its functional activity in vivo after the systemic administration of CytC-loaded PTNP at one of these doses (6 mg/kg body weight). As a result, the vascular architecture in adipose tissue was destroyed via targeted apoptosis of endothelial cells (Figure 2b). These findings provide further evidence to indicate that cytoplasmic delivery of CytC through intact PTNP inside the circulation is associated with adipose endothelial cell apoptosis in vivo.

The growth of adipose tissue is dependent on angiogenesis.4,5,6,7 Ablation or inhibition of vascular endothelial cells in adipose tissue by therapeutic applications has established as a promising strategy to control obesity.8,9,10,11,12 The intravenous administration of CytC-loaded PTNP prevented the percentage of increase in body weight for the HFD-fed mice in a dose-dependent manner (Figure 3). The weight reduction can be explained by a significant loss of SAT and EAT mass which is related to the reduction in their size and length (Figure 4a,b). Several antiangiogenic drugs (TNP-470, polyphenol compounds) prevented DIO with a decreased vasculature in adipose tissue. Ablation of vessels in adipose tissue promoted the loss of adipose tissue, as reported by the relation with adipocyte hypotrophy and adipocyte hypoplasia.8,9,10,11,12,31 In agreement, our present work also demonstrates that CytC prevents DIO in a dose-dependent manner via the targeted apoptosis of vascular endothelial cells in adipose tissue.

In developing a novel therapeutic protein, CytC for obesity, it may be feasible to investigate its effects on serum parameters which are related to obesity and the induction of toxicity that may limit the use of such protein. Treatment of the HFD-fed mice with CytC-loaded PTNP significantly reduced serum leptin levels in a dose-dependent manner. A significant suppression of serum leptin levels is correlated with adipose tissue hypoplasia, reduction in weight and size of fat depots32 as alluded to above (Figure 5a). The treated mice showed a tendency to have decreased serum levels of TNF-α (Figure 5b). TNF-α is a proinflammatory cytokine that is mainly secreted by macrophages during obesity.33 With the secretion, migration of several inflammatory cells such as macrophages and preadipocytes into adipose tissue is initiated and the subsequent result is the inflammation of adipose tissue that is highly associated with adipose tissue hypertrophy and insulin resistance.33,34 Thus, the treatment may also have an impact on a decrease in adipose tissue toxicity and an improvement in insulin sensitivity. The HFD-treated mice are known to have a tendency to a dyslipidemia characterized by a decrease in the levels of TG, total cholesterol, and free fatty acids (Figure 5c–e). However, the finding of no significant levels of several serum parameters such as TG, free fatty acids, and total cholesterol were present after treatment with the CytC-loaded PTNP system for 30 days suggests that further investigations with the aim of assessing the improvement of blood glucose and insulin action by the administration of the CytC-loaded PTNP system for a long term or to obese animals would be highly desirable. In addition, the evaluation of serum levels of liver enzymes, ALT in the CytC-treated mice by ALT assay indicated that no apparent hepatotoxicity develops as a result of treatment with CytC-loaded PTNP on day 30 (Figure 6).

In conclusion, the findings reported here show the first therapeutic value of a highly biodegradable protein, CytC even at very low dosage after cytoplasmic delivery through PTNP. With this strategy, CytC-loaded PTNP prevents DIO in C57BL/6 mice in a dose-dependent manner, which is associated with the targeted ablation of vascular endothelial cells via apoptosis in adipose tissue. Such a physiological protein has the potential to improve the efficacy of obesity therapy while reducing the toxicities associated with both the drug and the carrier in other mouse models of obesity.

Materials and Methods

Materials and animals. N-[(3-maleimide-1-oxopropyl) aminopropyl polyethylene glycolcarbamyl] distearoyl-sn-Glycero-3-phosphoethanolamine (Maleimide-PEG-DSPE) was bought from Nippon Oil and Fat (Tokyo, Japan). Peptide (Pep: GKGGRAKDGGC-NH2, purity: 93.6%, molecular weight: 1,004.15) was synthesized by Kurabo Industries, Osaka, Japan. CytC from bovine heart (purity: 97%, molecular weight: 12,327) was purchased from Sigma-Aldrich, St Louis, MO.

Five-week-old male wt C57BL/6J mice were purchased from SLC Japan (Shizuoka, Japan). All animals were acclimatized for 1 week before use. Animal experiments involved standard procedures approved by the Institutional Animal Care and Research Advisory Committee of Hokkaido University, Sapporo, Japan.

Nanoparticle preparation and characterization. We conjugated a prohibitin-targeted peptide (GKGGRAKDGGC-NH2) with maleimide-PEG5 kDa-DSPE as described previously.21 Egg yolk phosphatidylcholine and cholesterol-based nanoparticles in which a prohibitin-targeting peptide-conjugated PEG-lipid (Pep-PEG5 kDa-DSPE), PEG5 kDa-DSPE, PEG2 kDa-DSPE were incorporated on the surface with the following compositions: 1.25 mol% Pep-PEG5 kDa-DSPE and 1 mol% PEG2 kDa-DSPE of total lipids for PTNP, 1.25 mol% PEG5 kDa-DSPE and 1 mol% PEG2 kDa-DSPE of total lipids for NTNP, respectively, were prepared by the reverse phase evaporation method as previously described.21 In addition, rhodamine-DOPE (1 mol% of total lipids) or NBD-DOPE (1 mol% of total lipids) was attached on its surface and the aqueous phase with sulforhodamine (0.5 mmol/l) was loaded into nanoparticle. The sizes and zeta potentials of nanoparticles were measured by photon correlation spectroscopy on a MalvernZetasizer (Malvern instruments, Malvern, UK).

Confocal observation of double-labeled PTNP in vivo. Mice (6-weeks-old male wt C57BL/6J) were administered intravenously with double-labeled PTNP (lipid membrane: 1 mol% NBD-DOPE and aqueous phase: 0.5 mmol/l rhodamine in 10 mmol/l HEPES) to a total lipid dose of 0.1 mmol/kg. After 6 hours, the mice were anesthetized and as much blood as possible was removed by cardiac puncture. Tissues from the the adipose inguinal region, lungs, heart, kidney, and liver were collected and washed three times with Hank's Buffered Salt Solution and then cut into small pieces. The pieces, after washing with Hank's Buffered Salt Solution, were transferred to light-protected disposable microcentrifuge tubes (1.5 ml) containing Hank's Buffered Salt Solution (1 ml) and then placed on ice until use. The pieces that were transferred to glass-base dishes were viewed under a confocal laser scanning microscopy (A1; Nikon, Tokyo, Japan).

Meanwhile, blood was collected and centrifuged at 400g to remove blood cells. The plasma fractions that were obtained from the double-labeled PTNP system and the double-labeled non-targeted PEGylated nanoparticles (double-labeled NTNP) after transferring into glass-base dishes were observed by confocal microscopy (A1; Nikon).

Measurement of encapsulated CytC into nanoparticles. CytC was dissolved in HEPES buffer (pH 7.4) at the concentration of 1.2 mg/ml. The solution was applied as an aqueous phase to be encapsulated into nanoparticles. Then, free CytC (nonencapsulated) was washed away by carrying out two times ultracentrifugation for 30 minutes at 85,000g for each wash. Precipitation and bicinchoninic acid assays were performed for protein measurement without affecting interfering substances, as described previously with minor modification.35 Briefly, after washing the pellets with two ultracentrifugations as described above, the pellets containing various concentrations of CytC-loaded nanoparticles were dispersed in deionized water (0.1 ml). The final volume in each suspension (0.1 ml) was transferred to separate disposable microcentrifuge tubes (1.5 ml) with the addition of 1 µl sodium dodecyl sulfate (5% wt/vol) containing 0.1 N NaOH. The optical density (OD) of samples and controls (empty nanoparticle) as a blank was measured at 562 nm using a Beckman coulter DU-640 spectrophotometer (Beckman Coulter, Krefeld, Germany). We measured the RR of encapsulated CytC according to the formula: %RR = P1/P0 × 100, where P0 = OD of the total CytC used in nanoparticle (free + encapsulated), P1 = OD of CytC (encapsulated). In addition, we also measured the encapsulation of rhodamine, as described previously.22 The %RR of CytC and rhodamine using a 1.2 mg/ml of CytC and a 0.5 mmol/l solution of rhodamine was determined to be ~15.2 ± 0.3 and 2.8 ± 0.5%, respectively, and were chosen in all the proceeding experiments.

Moreover, we directly added the CytC solution (1.2 mg/ml) in a 1:1 (chloroform:diisopropyl ether) mixture (vol/vol) without lipids (egg yolk phosphatidylcholine and cholesterol) and the preparation was then probe sonicated at the same conditions as described previously21 to observe the impact on the integrity of CytC in this process. With or without probe sonication, the water phase (CytC) and the organic solvent phase were immiscible (Supplementary Figure S5a). In addition, after probe sonication, we measured the OD of the treated CytC solution, to confirm its active state. The absorbance of the CytC solution with or without treatment with an organic solvent was measured at 400 nm for the heme moiety and at 280 nm for the protein moiety due to the aromatic amino acids in the protein chain. As a result, the OD280 of the treated CytC solution was quite similar to that of the nontreated CytC solution, indicating that the CytC is not removed by the organic solvent (Supplementary Figure S5b). Furthermore, the OD400 of the treated CytC solution was decreased (around 18%) in comparison to the nontreated sample, indicating that the majority of the CytC protein appeared to retain its intactness (Supplementary Figure S5b). The ratio of the absorbance of heme and protein in the treated CytC after probe sonication was decreased but the majority of the CytC (around 90%) remained in its active state (Supplementary Figure S5c).

Quantitative evaluation of apoptosis activity through an in vitro apoptosis assay. Primary endothelial cells were isolated from murine adipose tissue (pcEC-IWAT) as described previously.21 pcEC-IWAT cells (5 × 104 cells/well) were cultured overnight in the presence of 1 ml EGM-2MV media (Lonza, Walkersville, MD) in 48-well plates. The cultured cells were incubated with 3, 6, and 12 µg/ml CytC-loaded PTNP after adding 0.5 ml EGM-2MV, whereas the finally applied lipid contents (0.65 µmol) were adjusted to that of the placebo (empty PTNP) and free CytC (3, 6, and 12 µg/ml) for 3 hours. After rinsing with 0.5 ml (−) phosphate-buffered saline, the cells were treated with 0.1 ml of lysis buffer in each well. The well plate was kept at −80 °C for 3 hours and the cells were detached from the wells with cell scrappers. The cell suspension was centrifuged for 2 minutes at 450g. The cell lysate, mixed with an equal volume (1:1) of caspase-Glo 9 reagent (G8212; Promega, Madison, WI), was incubated at room temperature for ~1 hour. The expression of caspase 9 was recorded as luminescence (relative luminescence unit) with a bio-instrument atto luminometer (AB-2250; ATTO, Osaka, Japan).

In addition, both pcEC-IWAT and NIH3T3 cells were also prepared as described above. These cells were treated with the CytC-PTNP system (6 µg/ml of CytC) and the physical mixture (empty PTNP (0.65 µmol) + CytC (6 µg/ml)), followed by incubation for 3 hours for induction of apoptosis. The expression of caspase 9 was recorded as luminescence (relative luminescence unit), as described above.

Characterization of apoptotic primary adipose endothelial cells by confocal microscopy. pcEC-IWAT cells were seeded on sterile 35 mm glass-base dishes in the presence of 1 ml EGM-2MV media. The cell density was 2 × 105 cells/well. The cells were incubated for 24 hours to 50% confluence and then incubated with 6 µg/ml of CytC-loaded PTNP while the finally applied lipid contents (0.65 µmol) were adjusted to that of the placebo (empty PTNP), free CytC (6 µg/ml), empty PTNP, and nontreated cells after adding 1 ml EGM-2MV for 6 hours. The cell nuclei were stained with Hoechst 33342 (final concentration: 2.5 µg/ml) for 15 minutes and the cells were observed by confocal laser scanning microscope with Plan Apo 20 x/NA objective lenses (Nikon).

Apoptosis assay in vivo. Mice (6-weeks-old male wt C57BL/6J) received a single dose of placebo (empty PTNP) or CytC-loaded PTNP (6 mg/kg body weight) intravenously via the tail vein or remain untreated. At 24 hours post-injection, the excised SAT was stained with a Green FLICA (fluorescent-labeled inhibitors of caspase) Caspase 9 Assay Kit (Immunochemistry Technology, Bloomington, MN) and Alexa647-labeled GS-IB4.

Moreover, mice (6-weeks-old male wt C57BL/6J) were intravenously injected with a single dose of a physical mixture (empty PTNP (0.1 mmol/kg) + CytC (6 mg/kg)) and the CytC-loaded PTNP system (6 mg/kg of CytC) or remain untreated. At 24 hours post-injection, tissues from several organs including inguinal region, heart, liver, spleen, lung, and kidney were collected, stained as described above, and then observed by confocal microscopy (A1; Nikon).

Antiobesity study. The HFD (58Y1 (5.10 kcal/g) composed of 34.9% fat, 23.1% protein, and 25.9% carbohydrates) and the ND (EQ 5L37 (3.12 kcal/g) with 4.5% fat, 25.0% protein, and 49.3% carbohydrates) were obtained from the TestDiet (Division of Land O'Lakes Purina Feed, Arden Hills, MN). CytC-loaded PTNP was prepared as described above at a concentration of 1.2 mg/ml CytC solution. We considered the RR of encapsulated CytC (15.2%) in the dose calculation. Six-weeks-old male wt C57BL/6J mice were randomly divided into five groups. Three groups with at least three mice per group along with an exposure of HFD were intravenously injected with 6, 1.2, and 0.25 mg/kg of CytC-loaded PTNP at 3-day intervals for 30 days whereas the other two groups (n = 3) were allowed on HFD and ND during this period, remaining untreated as a pair of controls. After the treatment, the mice (five groups) were killed and blood was drawn by cardiac puncture under anesthesia. The blood containing tubes were allowed to stand at room temperature for 3 hours and serum was then separated by centrifugation for 2 minutes at 300g. Whole serum portions were immediately stored at −20 °C for measurement of blood parameters. EAT and SAT were removed, images were obtained using a digital camera and the weights were determined.

In addition, we further measured the amount of food intake after the treatment of mice that were fed with HFD with the CytC-loaded PTNP system which was intravenously injected at the highest dose (6.0 mg/kg). The physical mixture (empty PTNP (0.1 mmol/kg) + CytC solution (6.0 mg/kg))-treated (HFD), nontreated (HFD), and nontreated (ND) were also taken as controls. The amount of food intake of the CytC-loaded PTNP-treated mice (n = 3) was measured every 24 hours during a 3-day internal. Moreover, while the food intake was measured, we also analyzed the blood glucose concentration of all groups during this feeding condition at 24-hour intervals, using Breeze2 (Bayer Healthcare, Osaka, Japan).

Measurement of blood parameters. The serum samples from the five groups were defrosted at room temperature and then analyzed. Leptin and TNF-α in serum were determined by ELISA assay (R&D systems, Minneapolis, MN). Serum TG were measured using a Triglyceride Quantification Kit (BioVision, Livingston, NJ). Serum-free fatty acid content, total cholesterol, and ALT were determined using a commercially available kit (Wako Pure Chemicals, Osaka, Japan).

Statistical analyses. All statistical analyses were performed using the JMP6 statistical package (SAS Institute, Cary, NC). Student's t-test was used to determine the significance of the difference between means of two groups. Dunnett's multiple comparison test was used to evaluate statistical significance between each group and HFD control group. A P value of <0.05 was considered to be significant.

SUPPLEMENTARY MATERIAL Figure S1. Cell type-specific apoptosis of the CytC-PTNP system. Figure S2. Confocal observation of plasma fractions for the confirmation of intact PTNP system in the circulation. Figure S3. In vivo apoptosis of vascular endothelial cells with a single dose of the CytC-PTNP. Figure S4. Measurement of cumulative energy intake and blood glucose level after treatment with the CytC-loaded PTNP system. Figure S5. Physicobiological properties of the CytC after treatment with organic solvent mixture.

Acknowledgments

This study was supported in part by grants from the Special Education and Research Expenses of the Ministry of Education, Culture, Sports, Science and Technology of Japan and by grants from Nagai Foundation, Tokyo, Japan. We thank Milton Feather for editing this manuscript. The authors declared no conflict of interest.

Supplementary Material

Supplementary Information
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