Research Article For reprint orders, please contact: [email protected]
Novel injectable thermosensitive hydrogels for delivering hyaluronic acid–doxorubicin nanocomplexes to locally treat tumors
Aim: A thermosensitive injectable hydrogel composed of nanocomplexes of doxorubicin and hyaluronic acid (HA) for the local treatment of cancer diseases was developed and characterized. Materials & methods: It was found that the addition of a divalent metal ion of Mg (MgCl2) was required to properly form HA–doxorubicin nanocomplexes, which were mixed with Pluronic® F127 (BASF, Germany) to form thermosensitive injectable hydrogels. Results: The DH700KMF-15 hydrogel resulted in efficient growth inhibition of C26 colon cancer cells and it selectively targeted the lymphatic system by the specific affinity of HA to the lymphatic system in vivo. Conclusion: The optimal formulations of DH700KMF-15 can increase the therapeutic efficacy of the local treatment of cancer with the potential for lymphotropic targeting to inhibit metastasis. Keywords: doxorubicin • hyaluronic acid • hydrogel • intratumoral • Pluronic F127
Research efforts on drug delivery systems have been devoted to developing viscoelastic hydrogels, which display resultant hydrogel properties suitably designed to ensure long residence times and controlled delivery of the active molecule(s) after local application [1] . Among them, the use of carriers for the release of therapeutically active ingredients based on polymeric solutions able to gel in situ in response to a physiological stimulus appears to be very practical and attractive. Thermosensitive amphiphilic triblock copolymers, namely poly(ethylene oxide)– poly(propylene oxide)–poly(ethylene oxide) (poloxamers), have been widely used in biomedical fields as a result of their ability to undergo phase-reverse thermal gelation. Current research on this type of thermosensitive hydrogel has mainly focused on poloxamer 407 (Pluronic® F127 [PF127]; BASF, Germany). Patients with metastatic cancer have lower 5-year survival rates than those with localized cancer, and the presence of even a few cancer cells in the lymph nodes of those patients was
10.2217/NNM.14.211 © 2014 Future Medicine Ltd
found as well. Therefore, treatment of lymphatic metastases in the early cancer phase plays an important role in patient survival. For this purpose, hyaluronic acid (HA) was reported to be an ideal carrier for localizing anticancer drugs to lymph nodes. HA is a polysaccharide formed of alternating d-glucuronic acid and N-acetyl d-glucosamine that is found in connective tissues of the body and primarily cleared by the lymphatic system (with a 12–72-h turnover half-life [2]). Several studies correlated increased HA synthesis and uptake with cancer progression and the metastatic potential [3] . Furthermore, invasive cancer cells overexpress CD44, the primary receptor for HA [4] , and are dependent on high concentrations of CD44-internalized HA for proliferation [5] . We think that anticancer drugs conjugated to or forming a complex with HA might be very efficacious against primary tumors and their lymphatic metastases [6] . For localized cancer therapy, intratumoral, peritumoreal and intravesical injections of thermal-sensitive hydrogels composed of PF127 were proposed [7–10] . PF127
Nanomedicine (Lond.) (Epub ahead of print)
Hua-Jing Jhan1, Jun-Jen Liu2, Ying-Chen Chen1, Der-Zen Liu3, Ming-Thau Sheu1,‡ & Hsiu-O Ho*,1,‡ 1 School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan, Republic of China 2 School of Medical Laboratory Science & Biotechnology, Taipei Medical University, Taipei, Taiwan, Republic of China 3 Graduate Institute of Biomedical Materials & Engineering, Taipei Medical University, Taipei, Taiwan, Republic of China *Author for correspondence: [email protected] ‡ Authors contributed equally
part of
ISSN 1743-5889
Research Article Jhan, Liu, Chen, Liu, Sheu & Ho has been widely used in biomedical fields as a result of its ability to undergo phase-reverse thermal gelation. This self-assembling process occurs through micellization, which is characterized by two key parameters: the critical micellization concentration and critical micellization temperature [11] . Injection of thermal-reversible hydrogels leads to the formation of a ‘depot’ at the site of administration that slowly and continuously releases the drug to the tumor and surrounding tissues. This kind of topical or injectable gel for physical targeting has additional advantages over passive or other actively targeted therapies in that it can deliver a drug throughout a tumor regardless of its vascular status, thus providing accurate dosing without systemic toxicity. The incorporation of HA as a targeting ligand carrier in poloxamer hydrogels can enhance the therapeutic efficacy against the primary tumor, with greater potential for inhibiting lymphatic metastases. In this study, we developed a novel injectable thermosensitive hydrogel based on PF127 and HA–doxorubicin (Dox) nanocomplexes for intratumoral administration to treat cancer diseases. It was found in our previous study [Ho H-O et al., Unpublished Data (2014)] that without a chemical cross-linker, the addition of divalent metal ions of Mg (MgCl2) was required to properly form HA–Dox nanocomplexes, which could be mixed with PF127 to form thermosensitive injectable hydrogels. Viscoelastic properties and gelation temperatures of the PF127–HA–Dox hydrogels were investigated by rheological analyses, and the structures of the hydrogels were characterized by scanning electron microscopy. The Dox release behavior and cell viability were studied in vitro. Moreover, the in vivo antitumor efficacy of PF127–HA–Dox hydrogels was examined in a tumor xenograft mouse model and lymphatropic targeting study. Materials & methods
Experimental animals
BALB/c mice (males, 5 weeks old) were obtained from BioLASCO Taiwan. All animal experiments were conducted in specific pathogen-free conditions with guidelines provided by the Taipei Medical University of Science and Technology for the care and use of animals for research purposes. Animals were housed in groups of five under a 12-h light/dark cycle, allowed food and water ad libitum and acclimatized for 1 week. The animal study was approved by the Laboratory Animal Center of Taipei Medical University (Taiwan). Preparation of Dox-unloaded & -loaded PF127–HA thermosensitive hydrogels
Thermosensitive HA–Dox hydrogels were prepared by a physical mixing method as follows: deionized water, 1% HA solution, 1 M MgCl2 solution and 30% PF127 were mixed together in a tube and put in an ice bath for 30 min. The mixture was then mechanically vortexed at 2000 rpm in an IKA® MS1 minishaker (IKA, Germany), and 10 mg/ml of the Dox solution was slowly added. After preparing the solution, we calculated the composition of each sample to reach the predetermined composition. Characterization of the hydrogels Rheological characterization
Rheological parameters were measured with a rheometer (HAAKE™ Rotation Rheometer RS-1; Thermo Scientific, MA, USA). Oscillatory shear stress and dynamic temperature ramp were the test methods used. The linear viscoelastic region (LVR) of a hydrogel was determined, and the elastic modulus (G’) values were compared under the same physical conditions while holding the temperature (25°C) and frequency (1 Hz) constant and increasing the stress strain from 0.01 to 100 Pa [12] . The rheological parameters that changed with temperature were measured at a fixed frequency of 1 Hz in a temperature range of 15–60°C and a heating rate of 1.5°C/min [13] .
Materials
Dox hydrochloride was purchased from Zhejiang Hisun Pharmaceutical (China). Hyaluronic acid (9 kDa) was a kind gift from Bloomage Freda Biopharm (China), while those of 50K, 700K, and 1800K were purchased from Foodchemifa (Japan). MgCl2, regenerated cellulose tubular membranes (Cellu Sep® T2, nominal molecular weight [MW] cut-off: 6000–8000; Membrane Filtration Products, TX, USA), daunorubicin HCl and all reagents were obtained from SigmaAldrich (MO, USA). PF127 (Kolliphor® P407) was purchased from BASF (Germany). Fetal bovine serum (FBS) was obtained from Biowest (France). All other chemicals were of analytical grade and were used without further purification.
10.2217/NNM.14.211 Nanomedicine (Lond.) (Epub ahead of print)
Sol–gel transition phase diagram
Solution (sol)–gel phase transition temperatures of the HA–Dox hydrogels and PF127 were determined using the test tube inversion method in a 15-ml test tube at a temperature increment of 2°C/step (temperature range: 5–85°C). The sol–gel transition temperature was determined by the flow or nonflow criterion over 1 min with the test tube inverted [14] . High-performance liquid chromatographic analysis of Dox
Samples were analyzed on a C18 column (250 × 4.6 mm, 5 μm, Discovery ®; SUPELCO, PA, USA). The mobile phase consisted of water:acetonitrile:methanol:acetic
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Injectable thermosensitive hydrogels for delivering hyaluronic acid–doxorubicin nanocomplexes
acid (650:250:100:2 v/v/v/v) adjusted to pH 3.6 with 2 N NaOH. The flow rate of the mobile phase was set to 1.0 ml/min. The fluorescence detector was operated at an excitation wavelength of 480 nm and an emission wavelength of 550 nm. The column oven temperature was maintained at 40°C. In total, 20 μl of sample was injected each time. In vitro drug release studies
Dox release profiles of the hydrogels were determined by a dialysis method. A total of 1 ml of the hydrogel containing 1 mg/ml of Dox was placed in a regenerated cellulose tubular membrane. The sample in the dialysis bag was immersed in 20 ml of prewarmed phosphate buffer at different pH values (pH 3.0–7.0) and placed in a water bath maintained at 37 ± 0.1°C with shaking at 100 rpm. In order to maintain sink conditions, at predetermined time intervals, all of the medium was withdrawn and replaced with an equal volume of fresh medium. All release studies were run in triplicate. The amount of Dox released was determined by high-performance liquid chromatography. Cell viability studies
C26 colon carcinoma cells and HT29 human colon cancer cells were cultured in RPMI-1640 with 10% FBS and 10% penicillin–streptomycin. MCF7 human breast cancer cells were cultured in Eagle’s minimum essential medium with 10% FBS and 10% penicillin–streptomycin. Cells were seeded at a density of 5 × 104 cells/well in 24-well plates. After 24 h of incubation at 37°C with 5% CO2, the medium was replaced with fresh medium containing various concentration of free Dox, DF-15, DMF-15 and DH9–1800K MF-15 hydrogels (the formulations are listed in Table 1) . After 24 h of incubation, cell survival was measured using a tetrazolium salt
Research Article
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A total of 500 μl of MTT (6 mg/ml) was added to each well for 4 h. The medium was removed and dried in an oven at 50°C for 2 h, and then 500 μl DMSO/ethanol (1:1, v/v) was added to dissolve any purple formazan crystals that had formed. Cell viability was measured by an ELISA reader, and the absorbance was set to 520 nm. All cell viability studies were run in triplicate. Tumor inhibition studies
A suspension of C26 cells (106 cells/100 μl) was injected subcutaneously into the right flank of BALB/c male mice using a syringe with a 26-G needle. After 7 days, the tumor size had reached 100–300 mm 3. C26 tumor-bearing mice were randomly divided into five groups (n = 4) and received the following treatments: group 1 mice were treated with sterile phosphate-buffered saline (PBS) as the control group; group 2 mice received free Dox (4 mg/kg in 0.1 ml); group 3 mice received DF-15; group 4 mice were treated with DMF-15; and group 5 mice received the DH700K MF-15 formulation. All of the mice received intratumoral injection administration. The mice were weighed, and the tumor size was measured using calipers for 10 days after the intratumoral injection. The tumor volume (V) was calculated by the formula: V = (length × [width] 2 )/2. The tumor volume fold change is estimated as (VtD – V0D)/V0D, where VtD is the volume of the tumor at time ‘t’ and V0D is the volume of the tumor at day 0. On day 10, the mice were sacrificed, and the tumor masses were harvested, weighed and photographed. The tumor inhibition rate was calculated using the equation: Tumor inhibition rate (%) = (Wc – Wt)/Wc, where Wc is the weight of the tumor in the control group and Wt is the weight of the tumor in the test formulation group [15] .
Table 1. Effect of the Puronic® F127 content on the status of hydrogels at 4, 25 and 37°C. Acronym
Dox (mg/ml)
HA700K (%)
MgCl2 (M) PF127 (%)
4°C
State at different temperatures 25°C
37°C
Free Dox
1
0
0
0
Solution
Solution
Solution
DF-15
1
0
0
15
Solution
Solution
Gel
DMF-15
1
0
0.1
15
Solution
Solution
Gel
DH700KMF-0
1
0.1
0.1
0
Gel
Gel
Solution
DH700KMF-5
1
0.1
0.1
5
Gel
Solution
Solution
DH700KMF-10
1
0.1
0.1
10
Solution
Solution
Solution
DH700KMF-15
1
0.1
0.1
15
Solution
Solution
Gel
DH700KMF-20
1
0.1
0.1
20
Solution
Gel
Gel
Dox: Doxorubicin; HA: Hyaluronic acid; PF127: Pluronic ® F127 (BASF, Germany).
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10.2217/NNM.14.211
Research Article Jhan, Liu, Chen, Liu, Sheu & Ho Pharmacokinetic studies
Fifteen BALB/c mice were used to investigate the effect of formulations on the pharmacokinetics of Dox after intravenous or subcutaneous administration. Mice were randomly divided into three groups (n = 5), and given a single dose of Dox (4 mg/kg in 0.1 ml). Group 1 mice were treated with an intravenous injection of free Dox; group 2 mice received a subcutaneous injection of free Dox; and group 3 mice received a subcutaneous injection of the DH700K MF-15 formulation. The plasma was immediately separated by centrifugation at 3500 rpm for 10 min. Plasma samples were frozen and maintained at -80°C until being analyzed. Plasma was extracted as described here: 5 μl of daunorubicin HCl as an internal standard (2 μg/ml) was added to a 20-μl plasma sample; it was then deproteinized with 200 μl of acetonitrile. After vortexing and centrifugation at 12,000 rpm at 4°C for 10 min, the supernatant was transferred to a clean tube and evaporated under a gentle stream of nitrogen gas at 40°C. The residue was reconstituted in 50 μl of the mobile phase for plasma sampling. After vortexing, a 20-μl sample was analyzed by high-performance liquid chromatography. In vivo biodistribution studies
Tumor-bearing mice were prepared by subcutaneously injecting a suspension of C26 cancer cells (106 cells/100 μl) into the right flank of BALB/c male mice. Seven days after the subcutaneous inoculation, mice in three groups of five animals each were given free Dox by an intravenous injection, free Dox by an intratumoral injection or DH700K MF-15 by an intratumoral injection at a single dose of 4 mg/kg. After 72 h, the mice were sacrificed by anesthesia and perfused with a PBS/EDTA solution to remove the blood from tissues. The heart, lungs, liver, spleen, kidneys and tumor were excised. All of them were weighed and stored at -20°C until being assayed for Dox. The process of tissue extraction used a homogenizer method. Before homogenization, a fivefold increased volume of the PBS/EDTA solution was added to each tissue. Tissues were homogenized using an SH-100 sample homogenizer (Kurabo Industries, Japan). A total of 20 μl of tissue homogenate was sampled, and the assay of the Dox concentration followed the same procedures as for the assay of plasma Dox concentrations described above. HA’s effect on the delivery of Dox to the lymphatic system
BALB/c mice were anesthetized with Zoletil® 50 (VIRBAC, Carros, France), and Dox formulations were injected subcutaneously into the second dorsal toe of the hind foot [16] . BALB/c mice were divided into four
10.2217/NNM.14.211 Nanomedicine (Lond.) (Epub ahead of print)
groups of 12 animals, each of which was given free Dox, DF-15, DMF-15 or DH700K MF-15 at a single injection of a 1-mg/kg dose in 25 μl via a subcutaneous injection into the second dorsal toe of the hind foot. At indicated times after the injection, mice were sacrificed by cervical dislocation, and the popliteal lymph nodes were immediately excised. The lymph nodes were stored at -20°C until being assayed for Dox. Lymph node extraction was followed by tissue extraction. Statistical analysis
Statistical analyses were performed by an analysis of variance. Differences were considered statistically significant if the p-value was < 0.05. Results Preparation of Dox loaded in HA–PF127 thermosensitive hydrogels
Preliminarily, the thermosensitivity expressed as the sol–gel–sol transition profile of the HA–PF127 hydrogels was examined at various ratios of HA to PF127, and typical results that are exemplified by using HA with an MW of 700K are illustrated in Figure 1A . This shows that the addition of HA700K to PF127 caused the sol–gel–sol temperature range to contract, with the solto-gel transition temperature (also referred to as the lower critical solution temperature) increasing and the gel-to-sol transition temperature (also referred to as the upper critical solution temperature) decreasing. Our preliminary study [Ho H-O et al., Unpublished Data (2014)] revealed that a certain molar ratio of Dox to Mg2+ was necessary to form HA–Dox nanocomplexes with no precipitation. Finally, the compositions and acronyms of the formulations tested for preparing themosensitive injectable hydrogels containing HA–Dox nanocomplexes in PF127 hydrogels are listed in Table 1. The optimal formulation was found to be DHMF-15, as it was in solution form at 25°C and transformed into the gel form at 37°C. The composition of DHMF-15 was 1.0 mg/ml Dox, 0.1% HA, 0.1 M MgCl2 and 15% PF127. Characterization of hydrogel Sol–gel–sol phase transition diagram
Sol–gel–sol phase transition diagrams for HA–Dox/Mg/ PF127 (DHMF) hydrogels were further examined with various MWs of HA (9, 50, 700 and 1800K), and the results are shown in Figure 1B. Compared with the PF127 hydrogel, DH50–1800K MF hydrogels displayed one sol–gel–sol transition curve in the higher PF127 concentration range and one gel–sol transition curve in the lower PF127 concentration range, while DH9K MF and PF127 hydrogels displayed only one sol–gel–sol transition curve in the higher PF127 concentration range.
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Injectable thermosensitive hydrogels for delivering hyaluronic acid–doxorubicin nanocomplexes
0.000% HA 0.025% HA 0.050% HA 0.075% HA
A
0.100% HA 0.125% HA 0.150% HA B 80
80
Temperature (°C)
Solution Temperature (°C)
Research Article
60
Gel
40
DH9KMF DH50KMF DH700KMF DH1800KMF PF127
60
Solution
40
Gel
20 Solution
Gel Solution
0
20 20
18
16
14
0
PF-127 concentration (%)
5
10
15
20
25
PF-127 concentration (%)
Figure 1. The sol-gel phase transition diagram. (A) HA700K /PF127 and (B) doxorubicin–HA9–1800K /MgCl2 with various PF127 concentrations. HA: Hyaluronic acid; PF127: Pluronic® F127 (BASF, Germany).
Rheological study of thermosensitive hydrogels
DH700K MF-15 within 24 h is 107.81, 79.54, 53.73, 28.01 and 14.28% at pH 3.0, 4.0, 5.0, 6.0 and 7.0, respectively. Regarding the influence of the pH value on the amount of drug released, DH700K MF-15 demonstrated that the release extent of Dox decreased with an increasing pH value of the dialysis media, with a plateau of release in the time range of 5–10 h.
The LVR profiles of DH9–1800K MF-15 were determined. The effect of the stress amplitude is shown in Figure 2A . The length of the LVR of DH9–1800K MF-15 determines the structural stability. A decrease in the slope of the curve indicates that structural breakdown has occurred as a consequence of the large deformations imposed. Figure 2A shows that the LVR of DH700K MF-15 was larger than the others. This demonstrates that HA700K is mostly suitable for producing injectable hydrogels [17] . The G’ and viscous modulus (G’’) of DH700K MF-15 at a frequency value of 1.0 Hz as a function of temperature are shown in Figure 2B. The gelation temperature was identified as the temperature at which the G’ and G’’ curves crossed each other. Figure 2B shows that G’ was higher than G’’ for temperatures above 36°C, but G’’ was higher than G’ when the temperature was below 36°C. This result demonstrates that the DH700K MF-15 hydrogel underwent a temperature-induced transition from a viscoelastic fluid to an elastic hydrogel when the temperature was over 36°C [13] . This indicates that gelation temperature of DH700K MF-15 is 36°C, which is close to body temperature and is suitable as a thermosensitive hydrogel for the following study.
The cellular cytotoxicity of HA–Dox hydrogels was assessed on C26 colon cancer, HT29 human colon cancer and MCF7 human breast cancer cell lines and the results are given in Table 2. The results demonstrated that a placebo (DH700K MF-15 containing no Dox) showed no cytotoxicity to C26, HT29 or MCF7 cells. Based on 50% inhibitory concentration values, it was found that the two hydrogel formulations containing no HA (DF-15 and DMF-15) and four hydrogel formulations containing various MWs of HA (DH9K MF-15, DH50K MF-15, DH700K MF-15 and DH1800K MF-15) were more effective at inhibiting the growth of C26, HT29 and MCF7 cells than free Dox, with the former (DF-15 and DMF-15) being slightly less effective than the latter (DH9K MF-15, DH50K MF-15, DH700K MF-15 and DH1800K MF-15) against C26 and MCF7 cells, but not HT29 cells.
In vitro Dox release
Tumor inhibition
The influence of pH values of the phosphate buffer on the drug release from DH700K MF-15 at 37°C is shown in Figure 3. The amount of Dox released from
Mice were randomized into five groups when the tumor size had reached 100 mm3. The five groups of mice received intratumoral injections of PBS, free
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Cell viability studies
10.2217/NNM.14.211
Research Article Jhan, Liu, Chen, Liu, Sheu & Ho
B 1000
10000
100
1000
10
100 G', G'' (Pa)
G' (Pa)
A
1 0.1
0.0001 0.001
1 0.1
DH50KMF-15 DH700KMF-15
0.001
G''
10
DH9KMF-15
0.01
G'
0.01
DH1800KMF-15
0.001 0.01
0.1
1
10
100
10
1000
20
30
40
50
60
70
Temperature (°C)
Strain (Pa)
Figure 2. Evaluation of rheological behavior. (A) Diagram of strain versus G’ values of DHMF-15 with different molecular weights of hyaluronic acid in a hydrogel system. (B) G’ and G’’ as a function of the temperature of the DH700KMF-15 formulation. The frequency of the applied oscillatory shear stress was 1 Hz. G’: Elastic modulus; G’’: Viscous modulus.
Dox, DF-15, DMF-15 and DH700K MF-15 hydrogels, respectively. Figure 4A shows changes in tumor volumes in C26-bearing mice after intratumoral administration, while Figure 4B delineates the tumor volume fold changes. In addition, we also monitored changes in body weight during the experiment, and the results demonstrated systemic toxicity (Figure 4C) . Furthermore, no symptoms of toxicity were observed after DH700K MF-15 was injected into the mice. After the mice had been sacrificed, the morphology of the tumors was examined, as shown is Figure 4D. The results indicate that the DH700K MF-15 hydrogel suppressed tumor growth. Tumor weights of the DH700K MF-15 hydrogel and free Dox groups were significantly reduced compared with the control group. The tumor inhibition rate of the DH700K MF-15
hydrogel was 91.35%, which was higher than those of free Dox at 69.24%, DF-15 at 70.77% and DMF-15 at 77.83% (Figure 4E) . Pharmacokinetics
The mean plasma concentration–time profiles of Dox in the blood after intravenous administration of free Dox or a subcutaneous injection of free Dox and DH700K MF-15 at a single dose of 4 mg/kg to mice are shown in Figure 5A, and the related pharmacokinetic parameters analyzed by WinNonlin® (Pharsight, MO, USA) software are given in Table 3. The area under the curve from 0 to 72 h (AUC0–72 h) of the intravenous free Dox (2204.23 ± 108.71 ng/h/ml) was higher than the subcutaneous free Dox (1558.15 ± 111.63 ng/h/ml) or subcutaneous DH700K MF-15 (1358.08 ± 176.67 ng/h/ml).
Table 2. 50% inhibitory concentration values of various hydrogel formulations against C26, HT29 and MCF7 cells. Formulations
50% inhibitory concentration values (μg/ml)
C26 cells
HT29 cells
MCF7 cells
Free doxorubicin
0.3204
0.8054
0.3936
DF-15
0.1101
0.6690
0.2347
DMF-15
0.1023
0.6828
0.2479
DH9KMF-15
0.0716
0.6715
0.1339
DH50KMF-15
0.0632
0.6779
0.1596
DHMF-15
DH700KMF-15
0.0654
0.6955
0.1773
DH1800KMF-15
0.0695
0.6658
0.1777
DHMF-15 placebo
None
None
None
10.2217/NNM.14.211 Nanomedicine (Lond.) (Epub ahead of print)
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Injectable thermosensitive hydrogels for delivering hyaluronic acid–doxorubicin nanocomplexes
By contrast, the peak concentration (Cmax) of the intravenous free Dox (318.15 ± 55.68 ng/ml) was lower than the subcutaneous free Dox (241.12 ± 88.78 ng/ml) or subcutaneous DH700K MF-15 (75.08 ± 6.98 ng/ml). Mice were intravenously administered with free Dox, given an intratumoral injection of free Dox or given an intratumoral injection of DH700K MF-15. After 72 h, we measured the amount of Dox that had accumulated in the heart, lungs, liver, spleen, kidneys and tumor. The amount of Dox with the DH700K MF-15 formation was higher at the tumor site compared with normal organs (Figure 5B) . Lymphatic system
In order to demonstrate that HA has ability to target and enhance the delivery of Dox to the lymphatic system, free Dox, DF-15, DMF-15 and DH700K MF-15 were injected subcutaneously into the second dorsal toe of the hind feet of BALB/c mice. Dox concentrations in the lymph nodes of mice at various times were then sampled and measured. The total amount of Dox that had accumulated in popliteal lymph nodes within 48 h (AUC0–48 h) was calculated and the results are given in Figure 6. However, although the AUC0–48 h values of the lymph nodes were 3005.30 ± 272.97 and 4550.86 ± 540.07 for DMF-15 and DH700K MF-15, respectively, which were lower than that for free Dox, a slower release from the optimal formulation of DH700K MF-15 was observed. Discussion In this study, we attempted to develop HA–PF127 thermosensitive hydrogels loaded with Dox as a novel injectable drug carrier for the potential local treatment of cancer. The presence of HA seemed to disturb the micellar packing and entanglements of PF127, leading to an increase in the sol-to-gel transition temperature, while the hydrophilicity of HA seemed to competitively dehydrate the poly(ethylene oxide) (PEO) shell layer in packed micelles, resulting in a decrease in the gel-to-sol transition temperature (Supplementary Material ; see online at: www.futuremedicine.com/doi/full/10.2217/ NNM.14.211). A preliminary study conducted in our laboratory [Ho H-O et al., Unpublished Data (2014)] showed that when Dox was mixed with HA–PF127 hydrogels in an aqueous solution, precipitation as an insoluble complex occurred. However, it was reported that in the presence of divalent metal ions, such as zinc or magnesium, the HA–doxycycline hydrogel was formed by polymerizing electrostatic interactions between HA and doxycycline molecules, followed by coordinated
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pH 4.0 pH 3.0
120 Doxorubicin release (%)
Biodistribution
pH 7.0 pH 6.0 pH 5.0
Research Article
100 80 60 40 20 0 0
5
10
15 20 Time (h)
25
30
Figure 3. Release profiles of doxorubicin from DH700KMF-15 in phosphate buffer at various pH values (pH 3.0–7.0; n = 3).
linking through the phenolic moieties of the immobilized doxycycline by divalent metal ion-mediated chelation [18] . Furthermore, it was also reported that HA degradation by oxygen-derived free radicals catalyzed by metal ions except manganese (II) showed little or no effect of degrading HA [19] . Therefore, since the chemical structure of Dox is similar to that of doxycycline, the formation of nanocomplexes of HA–Dox was first examined with the addition of various concentrations of MgCl2. The gel–sol transition curve in the lower PF127 concentration range of all DHMF formulations (except at the MW of 9K) exhibited a gel state at the low concentration of PF127, which was not observed for hydrogels employing only PF127 as the gelling polymer [20] . The gel–sol transition temperature for DHMF was maintained at approximately 30°C when the PF127 concentration was
Novel injectable thermosensitive hydrogels for delivering hyaluronic acid–doxorubicin nanocomplexes to locally treat tumors
Aim: A thermosensitive injectable hydrogel composed of nanocomplexes of doxorubicin and hyaluronic acid (HA) for the local treatment of cancer diseases was developed and characterized. Materials & methods: It was found that the addition of a divalent metal ion of Mg (MgCl2) was required to properly form HA–doxorubicin nanocomplexes, which were mixed with Pluronic® F127 (BASF, Germany) to form thermosensitive injectable hydrogels. Results: The DH700KMF-15 hydrogel resulted in efficient growth inhibition of C26 colon cancer cells and it selectively targeted the lymphatic system by the specific affinity of HA to the lymphatic system in vivo. Conclusion: The optimal formulations of DH700KMF-15 can increase the therapeutic efficacy of the local treatment of cancer with the potential for lymphotropic targeting to inhibit metastasis. Keywords: doxorubicin • hyaluronic acid • hydrogel • intratumoral • Pluronic F127
Research efforts on drug delivery systems have been devoted to developing viscoelastic hydrogels, which display resultant hydrogel properties suitably designed to ensure long residence times and controlled delivery of the active molecule(s) after local application [1] . Among them, the use of carriers for the release of therapeutically active ingredients based on polymeric solutions able to gel in situ in response to a physiological stimulus appears to be very practical and attractive. Thermosensitive amphiphilic triblock copolymers, namely poly(ethylene oxide)– poly(propylene oxide)–poly(ethylene oxide) (poloxamers), have been widely used in biomedical fields as a result of their ability to undergo phase-reverse thermal gelation. Current research on this type of thermosensitive hydrogel has mainly focused on poloxamer 407 (Pluronic® F127 [PF127]; BASF, Germany). Patients with metastatic cancer have lower 5-year survival rates than those with localized cancer, and the presence of even a few cancer cells in the lymph nodes of those patients was
10.2217/NNM.14.211 © 2014 Future Medicine Ltd
found as well. Therefore, treatment of lymphatic metastases in the early cancer phase plays an important role in patient survival. For this purpose, hyaluronic acid (HA) was reported to be an ideal carrier for localizing anticancer drugs to lymph nodes. HA is a polysaccharide formed of alternating d-glucuronic acid and N-acetyl d-glucosamine that is found in connective tissues of the body and primarily cleared by the lymphatic system (with a 12–72-h turnover half-life [2]). Several studies correlated increased HA synthesis and uptake with cancer progression and the metastatic potential [3] . Furthermore, invasive cancer cells overexpress CD44, the primary receptor for HA [4] , and are dependent on high concentrations of CD44-internalized HA for proliferation [5] . We think that anticancer drugs conjugated to or forming a complex with HA might be very efficacious against primary tumors and their lymphatic metastases [6] . For localized cancer therapy, intratumoral, peritumoreal and intravesical injections of thermal-sensitive hydrogels composed of PF127 were proposed [7–10] . PF127
Nanomedicine (Lond.) (Epub ahead of print)
Hua-Jing Jhan1, Jun-Jen Liu2, Ying-Chen Chen1, Der-Zen Liu3, Ming-Thau Sheu1,‡ & Hsiu-O Ho*,1,‡ 1 School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan, Republic of China 2 School of Medical Laboratory Science & Biotechnology, Taipei Medical University, Taipei, Taiwan, Republic of China 3 Graduate Institute of Biomedical Materials & Engineering, Taipei Medical University, Taipei, Taiwan, Republic of China *Author for correspondence: [email protected] ‡ Authors contributed equally
part of
ISSN 1743-5889
Research Article Jhan, Liu, Chen, Liu, Sheu & Ho has been widely used in biomedical fields as a result of its ability to undergo phase-reverse thermal gelation. This self-assembling process occurs through micellization, which is characterized by two key parameters: the critical micellization concentration and critical micellization temperature [11] . Injection of thermal-reversible hydrogels leads to the formation of a ‘depot’ at the site of administration that slowly and continuously releases the drug to the tumor and surrounding tissues. This kind of topical or injectable gel for physical targeting has additional advantages over passive or other actively targeted therapies in that it can deliver a drug throughout a tumor regardless of its vascular status, thus providing accurate dosing without systemic toxicity. The incorporation of HA as a targeting ligand carrier in poloxamer hydrogels can enhance the therapeutic efficacy against the primary tumor, with greater potential for inhibiting lymphatic metastases. In this study, we developed a novel injectable thermosensitive hydrogel based on PF127 and HA–doxorubicin (Dox) nanocomplexes for intratumoral administration to treat cancer diseases. It was found in our previous study [Ho H-O et al., Unpublished Data (2014)] that without a chemical cross-linker, the addition of divalent metal ions of Mg (MgCl2) was required to properly form HA–Dox nanocomplexes, which could be mixed with PF127 to form thermosensitive injectable hydrogels. Viscoelastic properties and gelation temperatures of the PF127–HA–Dox hydrogels were investigated by rheological analyses, and the structures of the hydrogels were characterized by scanning electron microscopy. The Dox release behavior and cell viability were studied in vitro. Moreover, the in vivo antitumor efficacy of PF127–HA–Dox hydrogels was examined in a tumor xenograft mouse model and lymphatropic targeting study. Materials & methods
Experimental animals
BALB/c mice (males, 5 weeks old) were obtained from BioLASCO Taiwan. All animal experiments were conducted in specific pathogen-free conditions with guidelines provided by the Taipei Medical University of Science and Technology for the care and use of animals for research purposes. Animals were housed in groups of five under a 12-h light/dark cycle, allowed food and water ad libitum and acclimatized for 1 week. The animal study was approved by the Laboratory Animal Center of Taipei Medical University (Taiwan). Preparation of Dox-unloaded & -loaded PF127–HA thermosensitive hydrogels
Thermosensitive HA–Dox hydrogels were prepared by a physical mixing method as follows: deionized water, 1% HA solution, 1 M MgCl2 solution and 30% PF127 were mixed together in a tube and put in an ice bath for 30 min. The mixture was then mechanically vortexed at 2000 rpm in an IKA® MS1 minishaker (IKA, Germany), and 10 mg/ml of the Dox solution was slowly added. After preparing the solution, we calculated the composition of each sample to reach the predetermined composition. Characterization of the hydrogels Rheological characterization
Rheological parameters were measured with a rheometer (HAAKE™ Rotation Rheometer RS-1; Thermo Scientific, MA, USA). Oscillatory shear stress and dynamic temperature ramp were the test methods used. The linear viscoelastic region (LVR) of a hydrogel was determined, and the elastic modulus (G’) values were compared under the same physical conditions while holding the temperature (25°C) and frequency (1 Hz) constant and increasing the stress strain from 0.01 to 100 Pa [12] . The rheological parameters that changed with temperature were measured at a fixed frequency of 1 Hz in a temperature range of 15–60°C and a heating rate of 1.5°C/min [13] .
Materials
Dox hydrochloride was purchased from Zhejiang Hisun Pharmaceutical (China). Hyaluronic acid (9 kDa) was a kind gift from Bloomage Freda Biopharm (China), while those of 50K, 700K, and 1800K were purchased from Foodchemifa (Japan). MgCl2, regenerated cellulose tubular membranes (Cellu Sep® T2, nominal molecular weight [MW] cut-off: 6000–8000; Membrane Filtration Products, TX, USA), daunorubicin HCl and all reagents were obtained from SigmaAldrich (MO, USA). PF127 (Kolliphor® P407) was purchased from BASF (Germany). Fetal bovine serum (FBS) was obtained from Biowest (France). All other chemicals were of analytical grade and were used without further purification.
10.2217/NNM.14.211 Nanomedicine (Lond.) (Epub ahead of print)
Sol–gel transition phase diagram
Solution (sol)–gel phase transition temperatures of the HA–Dox hydrogels and PF127 were determined using the test tube inversion method in a 15-ml test tube at a temperature increment of 2°C/step (temperature range: 5–85°C). The sol–gel transition temperature was determined by the flow or nonflow criterion over 1 min with the test tube inverted [14] . High-performance liquid chromatographic analysis of Dox
Samples were analyzed on a C18 column (250 × 4.6 mm, 5 μm, Discovery ®; SUPELCO, PA, USA). The mobile phase consisted of water:acetonitrile:methanol:acetic
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Injectable thermosensitive hydrogels for delivering hyaluronic acid–doxorubicin nanocomplexes
acid (650:250:100:2 v/v/v/v) adjusted to pH 3.6 with 2 N NaOH. The flow rate of the mobile phase was set to 1.0 ml/min. The fluorescence detector was operated at an excitation wavelength of 480 nm and an emission wavelength of 550 nm. The column oven temperature was maintained at 40°C. In total, 20 μl of sample was injected each time. In vitro drug release studies
Dox release profiles of the hydrogels were determined by a dialysis method. A total of 1 ml of the hydrogel containing 1 mg/ml of Dox was placed in a regenerated cellulose tubular membrane. The sample in the dialysis bag was immersed in 20 ml of prewarmed phosphate buffer at different pH values (pH 3.0–7.0) and placed in a water bath maintained at 37 ± 0.1°C with shaking at 100 rpm. In order to maintain sink conditions, at predetermined time intervals, all of the medium was withdrawn and replaced with an equal volume of fresh medium. All release studies were run in triplicate. The amount of Dox released was determined by high-performance liquid chromatography. Cell viability studies
C26 colon carcinoma cells and HT29 human colon cancer cells were cultured in RPMI-1640 with 10% FBS and 10% penicillin–streptomycin. MCF7 human breast cancer cells were cultured in Eagle’s minimum essential medium with 10% FBS and 10% penicillin–streptomycin. Cells were seeded at a density of 5 × 104 cells/well in 24-well plates. After 24 h of incubation at 37°C with 5% CO2, the medium was replaced with fresh medium containing various concentration of free Dox, DF-15, DMF-15 and DH9–1800K MF-15 hydrogels (the formulations are listed in Table 1) . After 24 h of incubation, cell survival was measured using a tetrazolium salt
Research Article
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A total of 500 μl of MTT (6 mg/ml) was added to each well for 4 h. The medium was removed and dried in an oven at 50°C for 2 h, and then 500 μl DMSO/ethanol (1:1, v/v) was added to dissolve any purple formazan crystals that had formed. Cell viability was measured by an ELISA reader, and the absorbance was set to 520 nm. All cell viability studies were run in triplicate. Tumor inhibition studies
A suspension of C26 cells (106 cells/100 μl) was injected subcutaneously into the right flank of BALB/c male mice using a syringe with a 26-G needle. After 7 days, the tumor size had reached 100–300 mm 3. C26 tumor-bearing mice were randomly divided into five groups (n = 4) and received the following treatments: group 1 mice were treated with sterile phosphate-buffered saline (PBS) as the control group; group 2 mice received free Dox (4 mg/kg in 0.1 ml); group 3 mice received DF-15; group 4 mice were treated with DMF-15; and group 5 mice received the DH700K MF-15 formulation. All of the mice received intratumoral injection administration. The mice were weighed, and the tumor size was measured using calipers for 10 days after the intratumoral injection. The tumor volume (V) was calculated by the formula: V = (length × [width] 2 )/2. The tumor volume fold change is estimated as (VtD – V0D)/V0D, where VtD is the volume of the tumor at time ‘t’ and V0D is the volume of the tumor at day 0. On day 10, the mice were sacrificed, and the tumor masses were harvested, weighed and photographed. The tumor inhibition rate was calculated using the equation: Tumor inhibition rate (%) = (Wc – Wt)/Wc, where Wc is the weight of the tumor in the control group and Wt is the weight of the tumor in the test formulation group [15] .
Table 1. Effect of the Puronic® F127 content on the status of hydrogels at 4, 25 and 37°C. Acronym
Dox (mg/ml)
HA700K (%)
MgCl2 (M) PF127 (%)
4°C
State at different temperatures 25°C
37°C
Free Dox
1
0
0
0
Solution
Solution
Solution
DF-15
1
0
0
15
Solution
Solution
Gel
DMF-15
1
0
0.1
15
Solution
Solution
Gel
DH700KMF-0
1
0.1
0.1
0
Gel
Gel
Solution
DH700KMF-5
1
0.1
0.1
5
Gel
Solution
Solution
DH700KMF-10
1
0.1
0.1
10
Solution
Solution
Solution
DH700KMF-15
1
0.1
0.1
15
Solution
Solution
Gel
DH700KMF-20
1
0.1
0.1
20
Solution
Gel
Gel
Dox: Doxorubicin; HA: Hyaluronic acid; PF127: Pluronic ® F127 (BASF, Germany).
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10.2217/NNM.14.211
Research Article Jhan, Liu, Chen, Liu, Sheu & Ho Pharmacokinetic studies
Fifteen BALB/c mice were used to investigate the effect of formulations on the pharmacokinetics of Dox after intravenous or subcutaneous administration. Mice were randomly divided into three groups (n = 5), and given a single dose of Dox (4 mg/kg in 0.1 ml). Group 1 mice were treated with an intravenous injection of free Dox; group 2 mice received a subcutaneous injection of free Dox; and group 3 mice received a subcutaneous injection of the DH700K MF-15 formulation. The plasma was immediately separated by centrifugation at 3500 rpm for 10 min. Plasma samples were frozen and maintained at -80°C until being analyzed. Plasma was extracted as described here: 5 μl of daunorubicin HCl as an internal standard (2 μg/ml) was added to a 20-μl plasma sample; it was then deproteinized with 200 μl of acetonitrile. After vortexing and centrifugation at 12,000 rpm at 4°C for 10 min, the supernatant was transferred to a clean tube and evaporated under a gentle stream of nitrogen gas at 40°C. The residue was reconstituted in 50 μl of the mobile phase for plasma sampling. After vortexing, a 20-μl sample was analyzed by high-performance liquid chromatography. In vivo biodistribution studies
Tumor-bearing mice were prepared by subcutaneously injecting a suspension of C26 cancer cells (106 cells/100 μl) into the right flank of BALB/c male mice. Seven days after the subcutaneous inoculation, mice in three groups of five animals each were given free Dox by an intravenous injection, free Dox by an intratumoral injection or DH700K MF-15 by an intratumoral injection at a single dose of 4 mg/kg. After 72 h, the mice were sacrificed by anesthesia and perfused with a PBS/EDTA solution to remove the blood from tissues. The heart, lungs, liver, spleen, kidneys and tumor were excised. All of them were weighed and stored at -20°C until being assayed for Dox. The process of tissue extraction used a homogenizer method. Before homogenization, a fivefold increased volume of the PBS/EDTA solution was added to each tissue. Tissues were homogenized using an SH-100 sample homogenizer (Kurabo Industries, Japan). A total of 20 μl of tissue homogenate was sampled, and the assay of the Dox concentration followed the same procedures as for the assay of plasma Dox concentrations described above. HA’s effect on the delivery of Dox to the lymphatic system
BALB/c mice were anesthetized with Zoletil® 50 (VIRBAC, Carros, France), and Dox formulations were injected subcutaneously into the second dorsal toe of the hind foot [16] . BALB/c mice were divided into four
10.2217/NNM.14.211 Nanomedicine (Lond.) (Epub ahead of print)
groups of 12 animals, each of which was given free Dox, DF-15, DMF-15 or DH700K MF-15 at a single injection of a 1-mg/kg dose in 25 μl via a subcutaneous injection into the second dorsal toe of the hind foot. At indicated times after the injection, mice were sacrificed by cervical dislocation, and the popliteal lymph nodes were immediately excised. The lymph nodes were stored at -20°C until being assayed for Dox. Lymph node extraction was followed by tissue extraction. Statistical analysis
Statistical analyses were performed by an analysis of variance. Differences were considered statistically significant if the p-value was < 0.05. Results Preparation of Dox loaded in HA–PF127 thermosensitive hydrogels
Preliminarily, the thermosensitivity expressed as the sol–gel–sol transition profile of the HA–PF127 hydrogels was examined at various ratios of HA to PF127, and typical results that are exemplified by using HA with an MW of 700K are illustrated in Figure 1A . This shows that the addition of HA700K to PF127 caused the sol–gel–sol temperature range to contract, with the solto-gel transition temperature (also referred to as the lower critical solution temperature) increasing and the gel-to-sol transition temperature (also referred to as the upper critical solution temperature) decreasing. Our preliminary study [Ho H-O et al., Unpublished Data (2014)] revealed that a certain molar ratio of Dox to Mg2+ was necessary to form HA–Dox nanocomplexes with no precipitation. Finally, the compositions and acronyms of the formulations tested for preparing themosensitive injectable hydrogels containing HA–Dox nanocomplexes in PF127 hydrogels are listed in Table 1. The optimal formulation was found to be DHMF-15, as it was in solution form at 25°C and transformed into the gel form at 37°C. The composition of DHMF-15 was 1.0 mg/ml Dox, 0.1% HA, 0.1 M MgCl2 and 15% PF127. Characterization of hydrogel Sol–gel–sol phase transition diagram
Sol–gel–sol phase transition diagrams for HA–Dox/Mg/ PF127 (DHMF) hydrogels were further examined with various MWs of HA (9, 50, 700 and 1800K), and the results are shown in Figure 1B. Compared with the PF127 hydrogel, DH50–1800K MF hydrogels displayed one sol–gel–sol transition curve in the higher PF127 concentration range and one gel–sol transition curve in the lower PF127 concentration range, while DH9K MF and PF127 hydrogels displayed only one sol–gel–sol transition curve in the higher PF127 concentration range.
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Injectable thermosensitive hydrogels for delivering hyaluronic acid–doxorubicin nanocomplexes
0.000% HA 0.025% HA 0.050% HA 0.075% HA
A
0.100% HA 0.125% HA 0.150% HA B 80
80
Temperature (°C)
Solution Temperature (°C)
Research Article
60
Gel
40
DH9KMF DH50KMF DH700KMF DH1800KMF PF127
60
Solution
40
Gel
20 Solution
Gel Solution
0
20 20
18
16
14
0
PF-127 concentration (%)
5
10
15
20
25
PF-127 concentration (%)
Figure 1. The sol-gel phase transition diagram. (A) HA700K /PF127 and (B) doxorubicin–HA9–1800K /MgCl2 with various PF127 concentrations. HA: Hyaluronic acid; PF127: Pluronic® F127 (BASF, Germany).
Rheological study of thermosensitive hydrogels
DH700K MF-15 within 24 h is 107.81, 79.54, 53.73, 28.01 and 14.28% at pH 3.0, 4.0, 5.0, 6.0 and 7.0, respectively. Regarding the influence of the pH value on the amount of drug released, DH700K MF-15 demonstrated that the release extent of Dox decreased with an increasing pH value of the dialysis media, with a plateau of release in the time range of 5–10 h.
The LVR profiles of DH9–1800K MF-15 were determined. The effect of the stress amplitude is shown in Figure 2A . The length of the LVR of DH9–1800K MF-15 determines the structural stability. A decrease in the slope of the curve indicates that structural breakdown has occurred as a consequence of the large deformations imposed. Figure 2A shows that the LVR of DH700K MF-15 was larger than the others. This demonstrates that HA700K is mostly suitable for producing injectable hydrogels [17] . The G’ and viscous modulus (G’’) of DH700K MF-15 at a frequency value of 1.0 Hz as a function of temperature are shown in Figure 2B. The gelation temperature was identified as the temperature at which the G’ and G’’ curves crossed each other. Figure 2B shows that G’ was higher than G’’ for temperatures above 36°C, but G’’ was higher than G’ when the temperature was below 36°C. This result demonstrates that the DH700K MF-15 hydrogel underwent a temperature-induced transition from a viscoelastic fluid to an elastic hydrogel when the temperature was over 36°C [13] . This indicates that gelation temperature of DH700K MF-15 is 36°C, which is close to body temperature and is suitable as a thermosensitive hydrogel for the following study.
The cellular cytotoxicity of HA–Dox hydrogels was assessed on C26 colon cancer, HT29 human colon cancer and MCF7 human breast cancer cell lines and the results are given in Table 2. The results demonstrated that a placebo (DH700K MF-15 containing no Dox) showed no cytotoxicity to C26, HT29 or MCF7 cells. Based on 50% inhibitory concentration values, it was found that the two hydrogel formulations containing no HA (DF-15 and DMF-15) and four hydrogel formulations containing various MWs of HA (DH9K MF-15, DH50K MF-15, DH700K MF-15 and DH1800K MF-15) were more effective at inhibiting the growth of C26, HT29 and MCF7 cells than free Dox, with the former (DF-15 and DMF-15) being slightly less effective than the latter (DH9K MF-15, DH50K MF-15, DH700K MF-15 and DH1800K MF-15) against C26 and MCF7 cells, but not HT29 cells.
In vitro Dox release
Tumor inhibition
The influence of pH values of the phosphate buffer on the drug release from DH700K MF-15 at 37°C is shown in Figure 3. The amount of Dox released from
Mice were randomized into five groups when the tumor size had reached 100 mm3. The five groups of mice received intratumoral injections of PBS, free
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Cell viability studies
10.2217/NNM.14.211
Research Article Jhan, Liu, Chen, Liu, Sheu & Ho
B 1000
10000
100
1000
10
100 G', G'' (Pa)
G' (Pa)
A
1 0.1
0.0001 0.001
1 0.1
DH50KMF-15 DH700KMF-15
0.001
G''
10
DH9KMF-15
0.01
G'
0.01
DH1800KMF-15
0.001 0.01
0.1
1
10
100
10
1000
20
30
40
50
60
70
Temperature (°C)
Strain (Pa)
Figure 2. Evaluation of rheological behavior. (A) Diagram of strain versus G’ values of DHMF-15 with different molecular weights of hyaluronic acid in a hydrogel system. (B) G’ and G’’ as a function of the temperature of the DH700KMF-15 formulation. The frequency of the applied oscillatory shear stress was 1 Hz. G’: Elastic modulus; G’’: Viscous modulus.
Dox, DF-15, DMF-15 and DH700K MF-15 hydrogels, respectively. Figure 4A shows changes in tumor volumes in C26-bearing mice after intratumoral administration, while Figure 4B delineates the tumor volume fold changes. In addition, we also monitored changes in body weight during the experiment, and the results demonstrated systemic toxicity (Figure 4C) . Furthermore, no symptoms of toxicity were observed after DH700K MF-15 was injected into the mice. After the mice had been sacrificed, the morphology of the tumors was examined, as shown is Figure 4D. The results indicate that the DH700K MF-15 hydrogel suppressed tumor growth. Tumor weights of the DH700K MF-15 hydrogel and free Dox groups were significantly reduced compared with the control group. The tumor inhibition rate of the DH700K MF-15
hydrogel was 91.35%, which was higher than those of free Dox at 69.24%, DF-15 at 70.77% and DMF-15 at 77.83% (Figure 4E) . Pharmacokinetics
The mean plasma concentration–time profiles of Dox in the blood after intravenous administration of free Dox or a subcutaneous injection of free Dox and DH700K MF-15 at a single dose of 4 mg/kg to mice are shown in Figure 5A, and the related pharmacokinetic parameters analyzed by WinNonlin® (Pharsight, MO, USA) software are given in Table 3. The area under the curve from 0 to 72 h (AUC0–72 h) of the intravenous free Dox (2204.23 ± 108.71 ng/h/ml) was higher than the subcutaneous free Dox (1558.15 ± 111.63 ng/h/ml) or subcutaneous DH700K MF-15 (1358.08 ± 176.67 ng/h/ml).
Table 2. 50% inhibitory concentration values of various hydrogel formulations against C26, HT29 and MCF7 cells. Formulations
50% inhibitory concentration values (μg/ml)
C26 cells
HT29 cells
MCF7 cells
Free doxorubicin
0.3204
0.8054
0.3936
DF-15
0.1101
0.6690
0.2347
DMF-15
0.1023
0.6828
0.2479
DH9KMF-15
0.0716
0.6715
0.1339
DH50KMF-15
0.0632
0.6779
0.1596
DHMF-15
DH700KMF-15
0.0654
0.6955
0.1773
DH1800KMF-15
0.0695
0.6658
0.1777
DHMF-15 placebo
None
None
None
10.2217/NNM.14.211 Nanomedicine (Lond.) (Epub ahead of print)
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Injectable thermosensitive hydrogels for delivering hyaluronic acid–doxorubicin nanocomplexes
By contrast, the peak concentration (Cmax) of the intravenous free Dox (318.15 ± 55.68 ng/ml) was lower than the subcutaneous free Dox (241.12 ± 88.78 ng/ml) or subcutaneous DH700K MF-15 (75.08 ± 6.98 ng/ml). Mice were intravenously administered with free Dox, given an intratumoral injection of free Dox or given an intratumoral injection of DH700K MF-15. After 72 h, we measured the amount of Dox that had accumulated in the heart, lungs, liver, spleen, kidneys and tumor. The amount of Dox with the DH700K MF-15 formation was higher at the tumor site compared with normal organs (Figure 5B) . Lymphatic system
In order to demonstrate that HA has ability to target and enhance the delivery of Dox to the lymphatic system, free Dox, DF-15, DMF-15 and DH700K MF-15 were injected subcutaneously into the second dorsal toe of the hind feet of BALB/c mice. Dox concentrations in the lymph nodes of mice at various times were then sampled and measured. The total amount of Dox that had accumulated in popliteal lymph nodes within 48 h (AUC0–48 h) was calculated and the results are given in Figure 6. However, although the AUC0–48 h values of the lymph nodes were 3005.30 ± 272.97 and 4550.86 ± 540.07 for DMF-15 and DH700K MF-15, respectively, which were lower than that for free Dox, a slower release from the optimal formulation of DH700K MF-15 was observed. Discussion In this study, we attempted to develop HA–PF127 thermosensitive hydrogels loaded with Dox as a novel injectable drug carrier for the potential local treatment of cancer. The presence of HA seemed to disturb the micellar packing and entanglements of PF127, leading to an increase in the sol-to-gel transition temperature, while the hydrophilicity of HA seemed to competitively dehydrate the poly(ethylene oxide) (PEO) shell layer in packed micelles, resulting in a decrease in the gel-to-sol transition temperature (Supplementary Material ; see online at: www.futuremedicine.com/doi/full/10.2217/ NNM.14.211). A preliminary study conducted in our laboratory [Ho H-O et al., Unpublished Data (2014)] showed that when Dox was mixed with HA–PF127 hydrogels in an aqueous solution, precipitation as an insoluble complex occurred. However, it was reported that in the presence of divalent metal ions, such as zinc or magnesium, the HA–doxycycline hydrogel was formed by polymerizing electrostatic interactions between HA and doxycycline molecules, followed by coordinated
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pH 4.0 pH 3.0
120 Doxorubicin release (%)
Biodistribution
pH 7.0 pH 6.0 pH 5.0
Research Article
100 80 60 40 20 0 0
5
10
15 20 Time (h)
25
30
Figure 3. Release profiles of doxorubicin from DH700KMF-15 in phosphate buffer at various pH values (pH 3.0–7.0; n = 3).
linking through the phenolic moieties of the immobilized doxycycline by divalent metal ion-mediated chelation [18] . Furthermore, it was also reported that HA degradation by oxygen-derived free radicals catalyzed by metal ions except manganese (II) showed little or no effect of degrading HA [19] . Therefore, since the chemical structure of Dox is similar to that of doxycycline, the formation of nanocomplexes of HA–Dox was first examined with the addition of various concentrations of MgCl2. The gel–sol transition curve in the lower PF127 concentration range of all DHMF formulations (except at the MW of 9K) exhibited a gel state at the low concentration of PF127, which was not observed for hydrogels employing only PF127 as the gelling polymer [20] . The gel–sol transition temperature for DHMF was maintained at approximately 30°C when the PF127 concentration was
