Contributed equally
Infantile hemangioma (IH) is a benign pediatric tumor, and rapid growth of IH can result in serious morbidity and even mortality. Only one drug Hemangeol™ (propranolol hydrochloride oral solution) has been approved for the treatment of IH, whereas patients suffer from its adverse effects and high frequency of administration. We have used urea, an organic compound and a normal body metabolite, in the treatment of IH for 20 years, and demonstrated that urea is an effective and well-tolerated treatment for IH. To reduce the daily administration of urea, we firstly utilized urea-loaded liposomes-in-microspheres (ULIM) as a novel topical controlled release system to realize the sustained release of urea. ULIM were fabricated from the encapsulation of urea-loaded liposomes in poly(lactic-
Infantile hemangioma (IH) affects approximately 4–10% of infants, and induces serious morbidity and mortality (
Urea, a normal body metabolite, is crucial for the metabolism of nitrogenous compounds (
To overcome the daily administration, a topical controlled delivery modality could be used for the treatment of IH. The topical administration could target diseases directly, and minimize unpredictable absorption and side-effects (
Liposomes, featured by their good biocompatibility and long
To reduce the daily administration of urea, we firstly utilized urea-loaded liposomes-in-microspheres (ULIM) as a novel topical controlled release system to realize the sustained release of urea. ULIM were developed from encapsulating urea-loaded liposomes in microspheres made of poly(lactic-co-glycolic acid)-b-poly(ethylene glycol)-b-poly(lactic-co-glycolic acid) copolymers. The characteristics, activity and mechanism against IH of ULIM were examined
Poly(lactic-
The human hemangioma endothelial cells (HemECs) derived from the IH of patients were isolated as previously described (
Urea-loaded liposomes were prepared by the reverse evaporization method as previously described (
Subsequently, urea-loaded liposomes-in-microspheres (ULIM) were prepared. After urea-loaded liposome solution (0.5 ml) was dispersed into an organic phase (20 mg PLGA-PEG-PLGA dissolved in 5 ml ethyl acetate) by vortex mixing, the W/O emulsion was injected drop-by-drop into 50 ml 2% PVA solution and underwent mechanical stirring (1,000 rpm for 5 min). The resultant W/O/W emulsion was poured into 2% PVA aqueous solution (450 ml). After then, the solution was stirred (500 rpm, 3 h) to evaporate the organic solvent. The final microspheres were obtained after filtration, washing and freeze-drying. Urea-loaded microspheres (UM) were prepared in the same way as ULIM, except that urea-loaded liposome solution was replaced with urea solution (0.5 ml, 4 mg/ml) as the water phase.
The following abbreviations are used: urea loaded liposomes (UL), urea-loaded microspheres (UM), and urea-loaded liposomes-in-microspheres (ULIM). Drug-free liposomes or microspheres are designated as blank liposomes or microspheres.
The size and zeta potential were analyzed by a Zetasizer Nano S (Malvern Instruments Ltd., Malvern, UK). The size and distribution of microspheres were tested by a Malvern Mastersizer 2000 particle size analyzer (Malvern Instruments).
The encapsulation efficacy and drug loading of urea in the formulations were determined as described below. After 0.2 ml of the liposome solution was dissolved in methanol, the clear solution was analyzed by high performance liquid chromatography (HPLC L-2000; Hitachi). Alternatively, 5 mg of microspheres were dissolved in 1 ml dichloromethane. After dichloromethane was evaporated, 1 ml methanol was added for HPLC analysis. A Hypersil NH2 column (250 × 4.6 mm, 5 µm) was equipped in the HPLC, and the mobile phase was acetonitrile:water (95:5, v/v). The flow rate was 1 ml/min. The detection wavelength was 190 nm. The column temperature was 25°C. The encapsulation efficacy of urea = the mass of encapsulated urea/the mass of total added urea × 100%. The drug loading of urea = the mass of encapsulated urea/the mass of liposomes or microspheres × 100%.
Two milliliters liposomes or 10 mg microspheres was transferred to a Spectra/Por® dialysis membrane (MWCO 1000). The sealed tube was put into a vial with 200 ml phosphate-buffered saline (PBS) (pH 7.4, with or without 10% FBS). The vial was put in a water bath at 37°C with stirring (100 rpm). A total of 2 ml of an aliquot of dialysate was taken out at different time-points. The concentration of urea was determined as described above.
HemECs (10,000/well) were seeded at subconfluency on a fibronectin-coated 96-well plate in EGM-2 with 10% FBS. Twelve hours later, the media were removed and the cells were treated with various concentrations of urea, liposomes or microspheres. After 72 or 120 h, the cell viability was determined using the CCK-8 kit according to the manual.
HemECs (200,000/well) were seeded on a fibronectin-coated 6-well plate in EGM-2 with 10% FBS. Twelve hours later, the medium was removed, and the cells were treated with urea (0, 10, 20 and 50 µg/ml) and incubated for a period of time (48, 72 or 96 h). For gene knockdown or overexpression, specific siRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or recombinant lentiviral vectors (Shanghai GenePharma Co., Ltd., Shanghai, China) were transfected to the cells and incubated for 48 h, before collection for analysis.
The cellular proteins were extracted, and separated by SDS-polyacrylamide gel electrophoresis. After the proteins were transferred onto polyvinylidene fluoride membranes, the membrane were incubated with the primary antibodies including anti-human HIF-1α or VEGF and horseradish peroxidase conjugated to goat anti-mouse IgG as the secondary antibody (Santa Cruz Biotechnology). The GAPDH antibody (Santa Cruz Biotechnology) was used as the internal control antibody. The bands were detected with the Enhanced Chemiluminiscence kit (GE Healthcare) and visualized with the ChemiDoc XRS system (Bio-Rad Laboratories, Inc., Hercules, CA, USA).
HemECs (200,000/well) were seeded on a fibronectin-coated 6-well plate in EGM-2 with 10% FBS. Twelve hours later, the medium was removed and the cells were treated with various concentrations of the drugs. After a period of time, the VEGF-A concentration in the cellular supernatant was measured by VEGF-A ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. In brief, 200 µl of the sample was added to the plate and incubated for 2 h. The sample was aspirated and washed. Afterwards, 200 µl of conjugate was added and incubated for 2 h. Finally, the substrate solution and stop solution was added sequentially. The absorbance was measured at 450/540 nm using a BioTek ELx800 Universal microplate reader.
The mice were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences (Shanghai, China). All procedures were approved by the Committee on Animals of the Second Military Medical University (Shanghai, China) and all procedures were performed in accordance with the guidelines of the Committee on Animals of the Second Military Medical University (Shanghai, China).
A xenograft mouse model of IH was used to study the effects of urea on IH
Data were analyzed with the software SPSS 13.0 (SPSS, Inc., Chicago, IL, USA). A direct comparison between two groups was performed with the Students non-paired t-test. One-way ANOVA with the Dunnetts or Newman Keuls post-test was used to compare the means of three or more groups. A P<0.05 was considered statistically significant: *P<0.05; **P<0.01; ***P<0.001; n.s. represents not significant (P>0.05).
As described in
Although the hydrophilic head groups protect liposomes, the liposomes could be readily damaged by the organic solvent used for the preparation of ULIM. Thus, chitosan was used to
The drug release profiles of UL, UM and ULIM were evaluated in PBS and PBS with 10% FBS. In PBS, UL showed a quick urea release (45% of urea was released at day 1, and 85% at day 4) (
Similar results were obtained in the urea release in PBS with 10% FBS. UL showed a quick urea release (59% of urea was released at day 1 and 95% at day 4) (
Thus, the urea release was significantly reduced in ULIM compared with UL and UM, suggesting that encapsulation of urea-loaded liposomes-in-microspheres significantly retard the release of urea from liposomes or microspheres.
To assess the biocompatibility of our prepared formulations, we evaluated the cytotoxicity of the blank liposomes, microspheres and LIM. As shown in
On the contrary, urea, UL, UM and ULIM showed a dose-dependent cytotoxicity towards HemECs (
The heparin-binding growth factor VEGF-A is able to induce angiogenesis (
Hypoxia-inducible factor-1α (HIF-1α) and VEGF-A are critical factors in promoting angiogenesis (
The therapeutic effect of the various formulations was examined in mice bearing subcutaneous hemangioma. As shown in
The hemangioma was weighed at the endpoint (
The toxicity of all treatments was measured by observing any physical or behavioral changes post treatment and by monitoring the weight of mice. In the urea-treated group, skin irritation, as reflected by the red and swollen skin, was observed in some mice, but all cases of skin irritation
The hemangiomas excised from the mice were stained with hematoxylin and eosin (H&E) (
IH is a benign pediatric tumor, and rapid growth of IH can result in serious morbidity and even mortality. We demonstrated that urea is an effective and well-tolerated treatment for IH (
The selection of an anti-hemangioma is critically important for the superior activity of our prepared ULIM. Urea is an organic compound and a normal body metabolite, and is widely used in various skin diseases (
Changing the route of administration could enhance the therapeutic efficacy and reduce the side-effects of drugs. To overcome the high frequency of urea administration, we developed a practical sustained release system defined as ULIM to release urea. The data presented here confirmed that ULIM showed a significantly slower release of urea, compared with UL and UM, as reflected by the fact that it took 4, 10 and 20 days for UL, UM and ULIM to release >80% of urea, respectively. Fen
Although ULIM were less efficient in the inhibition of the proliferation of HemECs and VEGF expression than urea, UL and UM
We investigated the effect of urea on the expression of VEGF-A and HIF-1α which promote angiogenesis (
The safety of a drug delivery systems is important in the clinic (
Our results elucidated the mechanism of the anti-hemangioma activity of ULIM (
In conclusion, we demonstrated that urea is an effective and well-tolerated treatment of IH, whereas its frequent administration reduces the compliance of patients. We hereby firstly utilized ULIM as a topical controlled release system to realize the sustained release of urea. ULIM have been demonstrated to show sustained release of urea, achieving superior therapeutic efficacy compared with urea and propranolol, and significantly reducing the administration frequency of urea. Moreover, the safety of ULIM is rather promising. Thus, our findings show that ULIM is a promising treatment for IH.
The present study was supported by the Henan Medical Provincial Science and Technology Plan Project (project number: 142102310080 and 201702202).
Preparation of urea-loaded liposomes and liposomes-in-microspheres. After specific amounts of lipids (HSPC and cholesterol) were dissolved in diethyl ether, urea solution was added to the lipid solution. The mixture was sonicated to stable W/O emulsion. The diethyl ether was removed by reduction vaporization to form multilamellar liposomes (MLL). Unilamellar liposomes (ULL) were obtained by extruding the MLL with a membrane extruder. Afterwards, the liposomes were coated with chitosan. The liposomes were dispersed into an organic phase consisting of PLGA-PEG-PLGA dissolved in ethyl acetate, resulting in a W/O emulsion. The W/O emulsion was injected drop-by-drop into PVA aqueous solution, and the resultant W/O/W emulsion was lyophilized to obtain urea-loaded liposomes-in-microspheres.
Drug release of liposomes and microspheres. A total of 2 ml of liposome solution or 10 mg microspheres were transferred to a dialysis membrane. The sealed tube was then introduced into a vial containing PBS with or without 10% FBS. The vial was secured in a water bath at 37°C with stirring. At predetermined time intervals, the amount of urea in the dialysate was determined by HPLC. (A) The
Dose-dependent cytotoxicity in HemECs at 72 and 120 h. The cytotoxicity of (A) blank liposomes and (B) blank microspheres and LIM towards HemECs at 72 h. The cytotoxicity of urea-loaded formulations towards HemECs at (C) 72 h and (D) 120 h. The cell viability was evaluated by the CCK-8 assay. Data are expressed as mean ± SD (n=3). ULIM, urea-loaded liposomes-in-microspheres; UL, urea-loaded liposomes; UM, urea-loaded microspheres; LIM, liposomes-in-microspheres.
VEGF-A expression level in HemECs after treatment. After HemECs were seeded on a fibronectin-coated 6-well plate, the cells were treated with various concentrations of urea-loaded formulations for 72 or 120 h. The VEGF-A concentration of the cellular supernatant was measured by VEGF-A ELISA kits. The relative protein level was expressed as the percentage of the protein of the treated groups relative to the untreated group. The relative protein level of the urea-treated group was compared with other groups by one-way ANOVA with the Dunnett's post-test. *P<0.05; **P<0.01; ***P<0.001. Data are expressed as mean ± SD (n=3). UL, urea-loaded liposomes; UM, urea-loaded microspheres; ULIM, urea-loaded liposomes-in-microspheres.
Effect of urea on the expression of HIF-1α in HemECs
Therapeutic effect of urea in mice bearing subcutaneous IH xenografts. When the hemangiomas had reached ~25 mm3 in size (day 0), mice were treated with single intratumoral injections of either formulation (UL, UM and ULIM, 2 mg urea/kg), free urea (2 mg urea/kg) or blank LIM (80 mg/kg). Propranolol was administrated orally (2 mg/kg, daily) for 30 days. Treatments were carried out on days 0, 5, 10, 15, 20, 25 and 30 (indicated by black arrows). (A) The emangioma growth curve. (B) Hemangioma volume at the end point (day 35). (C) The excised tumors were weighed at the end point. The tumor volume or weight of the ULIM-treated group was compared with that of other groups by one-way ANOVA with the Dunnett's post-test. *P<0.05; **P<0.01; ***P<0.001. (D) The weight change of the mice during the treatment. The body weight of the mice was monitored once every five days. Data are expressed as mean ± SD (n=8). UL, urea-loaded liposomes; UM, urea-loaded microspheres; ULIM, urea-loaded liposomes-in-microspheres.
The therapeutic effect of urea in mice bearing subcutaneous IH xenografts, as reflected by the H&E staining of sections and microvessel density (MVD) analysis of the sections. On day 35, the mice were euthanized. The hemangiomas were collected, fixed and embedded in paraffin for histological analysis. (A) H&E staining of sections. Black arrows indicated lumens with red blood cells. Scale bars represent 100 µm. (B) MVD quantification of hemangioma. Lumens with red blood cells (A, arrows) were counted. Data are expressed as mean ± SD (n=8). *P<0.05; **P<0.01; ***P<0.001. UL, urea-loaded liposomes; UM, urea-loaded microspheres; ULIM, urea-loaded liposomes-in-microspheres.
Mechanism underlying the anti-hemangioma activity of ULIM. After being released from the ULIM, urea inhibits the proliferation of HemECs, and the production of angiogenesis factors including VEGF-A and HIF-1α, resulting in inhibition of IH growth. ULIM, urea-loaded liposomes-in-microspheres.
Characterization of nanoparticles and microspheres.
Size (nm/µm) | Zeta potential (mV) | PDI | EE (%) | Drug loading (%) | |
---|---|---|---|---|---|
UL | 186.3±24.7 nm | −6.8±2.4 | 0.16±0.04 | 31.7±8.7 | 10.3±3.1 |
ULIM | 62.4±21.3 µm | – | – | 51.5±8.6 | 2.5±0.5 |
UM | 53.8±25.9 µm | – | – | 55.2±4.2 | 3.4±2.7 |
Data are expressed as mean ± SD (n=3) from three independent samples. UL, urea-loaded liposomes; UM, urea-loaded microspheres; ULIM, urea-loaded liposomes-in-microspheres. PDI, polydispersity; EE, encapsulation efficacy.
IC50 values in HemECs following 72 and 120 h of treatment.
IC50, µg/ml | 72 h | 120 h |
---|---|---|
Urea | 168±29 | 98±21 |
UL | 218±36 | 133±32 |
UM | 502±52 | 389±76 |
ULIM | 1238±252 | 631±139 |
Data are expressed as mean ± SD (n=3). UL, urea-loaded liposomes; UM, urea-loaded microspheres; ULIM, urea-loaded liposomes-in-microspheres.