Preliminary pharmacokinetics of PEGylated oxaliplatin polylactic acid nanoparticles in rabbits and tumor-bearing mice

Abstract

To testify the targeting effect of PEGylated Oxaliplatin polylactic acid (OP-PEG-PLA) nanoparticles (NPs), we studied drug concentration in rabbit plasma and tissue distribution in tumor-bearing mice. Concentration of nanoparticle colloidal solution was performed with dialysis. Qualities of enriched NPs were characterized by particle size and drug content. OP concentration in samples was detected using ICP-MS. Compared to OP solution groups, OP concentration of NPs groups increased in the tumor (p < 0.05) and decreased in the kidney and heart (p < 0.05). Compared to OP-PLA NPs groups, OP concentration of OP-PEG-PLA NPs groups increased in the tumor and decreased in the liver and lung (p < 0.05). The concentrated OP-PEG-PLA NPs are good in clinical application and tumor delivery.

Keywords::

• nanoparticle
• oxaliplatin
• nanoparticle
• pharmacokinetics
• tumor

Introduction

Compared to traditional medicine, nanoparticles (NPs) have advantages in medicine release, treatment, targeting and other aspects. The comparatively smaller particle size makes NPs more penetrative than traditional ones. Enhanced permeation and retention effect (EPR) of tumor leads to more effective entrance of NPs into tumors.

Biodegradable nanoparticle was put forward in 1979 (Couvreur et al. 1979). Polylactic acid (PLA), which can produce carbon dioxide and water in physical environment, is deemed as a relatively safe material for medical uses. PLA hinders the therapeutic effect of NPs because it produces a hydrophobic shell that facilitates the phagocytosis of NPs into mononuclear phagocytes (Brigger et al. 2002). However, polyethylene glycol (PEG) has so good water solubility and biocompatibility that it can impede the uptake of NPs by mononuclear phagocytes (Kim et al. 2003, Gref et al. 2000). Consequently, we chose PEGylated PLA to modify NPs (Cui et al. 2013) and prepared PEGylated Oxaliplatin polylactic acid (OP-PEG-PLA) NPs.

Nanoparticle colloidal solution of a high concentration reduces dosing frequency and improves patient's compliance. Accordingly, we concentrated the colloidal solution by dialysis and performed in vivo researches. Firstly, we analyzed OP concentration in rabbit plasma to obtain pharmacokinetic parameters of the nanoparticle. Secondly, we detected drug distribution in organs of tumor-bearing mice to determine the targeting effect of the nanoparticle.

Materials and instruments

Oxaliplatin (98.5%), Qilu pharmaceutical Co., Ltd; OP-PLA NPs (self-made, 76.45 μg/g); OP-PEG-PLA NPs (self-made, 75.96 μg/g), PEG 4000, Sinopharm Chemical Regent co., Ltd; Dialysis bag (MW cutoff 3,500), Shanghai Yuanye Bio-Technology Co., Ltd; Single element labeling solution of Pt and ¹⁸⁷Re are provided by National Research Center for Certified Reference Materials, China; Kunming Mice, 25 ± 5 g, Animals certificate: SCXK(E) 20120022; New Zealand Rabbits, 1.5 ± 0.5kg, animals certificate: SCXK(E) 20120076; laser particle size analyzer (LB550), HORIBA; HPLC (LC-20A), Shimadzu; ICP-MS (ICP-MS7500A), Agilent, America.

Methods

Concentration of NPs colloidal solutions

Dialysis

The nanoparticle colloidal solutions prepared by previous experiment (Couvreur et al. 1979) using dialysis with PEG4000 as concentrating agent (Vauthier et al. 2008). Added 3 g of OP-PEG-PLA NPs to a dialysis bag (MW cut-off 3,500), then immersed the bag in PEG4000 solution (9 ml, 30%) for 2 h. OP-PLA NPs were treated in the same way as a control.

Measurement of particle sizes and OP contents

Particle size was measured with a particle size analyzer. OP content in concentrated solution was determined by HPLC.

Drug metabolism in plasma of rabbits

Grouping and administration

Nine rabbits were randomly divided into three groups. After 12 h of fasting, OP-PLA NPs, OP-PEG-PLA NPs and OP solutions were administered using intravenous injection (3 mg/kg) through ear vein to the three groups of rabbits, respectively. All rabbits were permitted to eat and drink as much as they wanted after injection.

Sample collection

From the rabbit heart 2 ml of blood is extracted at 0, 0.5, 1, 2, 4, 8, 12, 24 and 48 h after administration and then the blood is transferred into tubes containing heparin. Plasma is separated with centrifugation and stored at − 20°C.

Detection

One millilitre of plasma sample or 1 g of organ sample was added into crucible accurately. The crucible was placed on an electric furnace to carbonize it until all plasma turned to smoke. Those samples were made into ashes in a muffle furnace at 650°C for 2 h. If dark residue in a sample was found, 0.5 ml of nitric acid was added and the sample was made into ashes again until all such residue disappears. One millilitre of aqua regia (1:1) and 9 ml of distilled water were added to the sample and heated on the electric furnace for 10 min. Water was added to the crucible until it reaches 10 ml. Finally, the sample with ICP-MS was determined. The reference substance of reducing solution was prepared. OP concentration in samples was calculated as follows:

Drug distribution in tumor-bearing mice

Establishment of Sacromaia 180-bearing mice models

Lower quadrat of the Sacromaia 180 (S180)-bearing mice was disinfected, and 50 μl of ascite from the lower quadrat was extracted. 450 μl of normal saline (NS) was added to the ascites uniformly, the cells were counted on a cell-counting chamber and then the concentration of the cells was adjusted to 1 × 10⁷/ml. Then 0.2 ml of cell suspension was injected into the armpits of mice. The operation was repeated everyday until the tumor grew to 100–150mm³.

Grouping and administration

Twenty-seven tumor-bearing mice were assigned to three groups randomly. After 12 h of fasting, OP-PLA NPs, OP-PEG-PLA NPs and OP solutions were administered using intravenous injection (3mg/kg) through tail vein to the three groups of mice, respectively. All mice were permitted to eat and drink as much as they wanted after injection.

Sample collections and disposes

Half an hour after administration, three mice were sacrificed randomly from each group. Their brains, hearts, livers, spleens, lungs, kidneys and tumors were removed . They were treated with physiological sodium chloride solution, and then 1 g of each organ was transferred into an eppendorf tube. They were dried for 5 h at 55°C and stored at − 20°C. The operation was repeated at 4 and 12 h after the administration.

Statistical analysis

Statistical significance between groups during analysis was compared using SNK method with SPSS19.0. A p value of less than 0.05 was considered as an evidence of a significant difference.

Results

Character of concentrated NPs

Particle size of the concentrated NPs

Mean particle sizes of concentrated OP-PEG-PLA NPs and OP-PLA NPs are 154.6 ± 14nm and 135 ± 13nm, respectively. And mean particle size of non-concentrated OP-PEG-PLA NPs and OP-PLA NPs are 138nm and 120nm, respectively. Compared to non-concentrated NPs, the concentrated ones had a slightly bigger particle size. As is shown in Figures 1 and 2, size distribution of concentrated NPs covered wider than that of non-concentrated ones.

Figure 1. Particle size distribution of concentrated OP-PLA NPs.

Figure 2. Particle size distribution of concentrated OP-PEG-PLA NPs.

Determination of OP concentration

OP concentration in NPs (P) was calculated with computational formula: , where M₀ is the weight of concentrated NPs, M₁ is OP content in primary nanoparticle solution and M₂ is OP content in dialysate after dialysis. The results are shown in Tables I and II.

Table I. OP concentrations of concentrated OP-PLA NPs.

Sample No.	1	2	3	Mean ± s
M0 (μg)	0.2921 × 10^6	0.3011 × 10^6	0.2820 × 10^6	(0.2917 ± 0.01)× 10^6
M1 (μg)	204.05	204.05	204.05	204.05
M2 (μg)	74.65	78.36	79.43	77.48 ± 2.51
P (μg/mg)	0.4430	0.4174	0.4419	0.43 ± 0.02

Table II. OP concentrations of concentrated OP-PEG-PLA NPs.

Sample No.	1	2	3	Mean ± s
M0 (μg)	0.2932 × 10^6	0.2923 × 10^6	0.2843 × 10^6	(0.2899 ± 0.01) × 10^6
M1 (μg)	230.88	230.88	230.88	230.88
M2 (μg)	75.54	77.45	78.54	77.18 ± 1.52
P (μg/mg)	0.5298	0.5249	0.5358	0.53 ± 0.01

OP content in rabbit plasmas

OP concentration in rabbit plasma was determined in the way mentioned above. The concentration time curve is shown in Figure 3.

Figure 3. Concentration–time curve of OP in rabbit plasmas.

The results indicated that the concentration time curve of OP in rabbits conforms to two compartment model according to statistics with 3p87 (Table III).

Table III. The pharmacokinetic parameters of OP solution, OP-PLA NPs and OP-PEG-PLA NPs in rabbit plasma.

Parameter	Unit	OP sol	OP-PLA NPs	OP-PEG-PLA NPs
A	μg/ml	37.26	35.79	32.89
α	1/h	1.20	0.68	0.60
B	μg/ml	5.35	6.97	8.35
β	1/h	0.035	0.040	0.044
V (c)	L/kg	0.070	0.071	0.073
T1/2α	h	0.578	1.016	1.168
T1/2β	h	15.64	17.18	20.00
K21	1/h	0.18	0.15	0.16
K10	1/h	0.23	0.19	0.17
K12	1/h	0.82	0.39	0.31
AUC	μg/ml·h	185.48	225.22	243.93
CL(s)	mg/kg/h/(μg/ml)	0.0162	0.0133	0.0123

Results of OP distribution in tumor-bearing mice

At half an hour after administration, OP content in tumor increased significantly (p < 0.05) in the mice given OP solution administration (see Figure 4). There was no remarkable difference among members in the same group. As to the group given OP-PLA NPs, OP concentrations in the liver, spleen and lungs were greater than other organs (p < 0.05). Moreover, OP concentration in spleen of mice given OP-PEG-PLA NPs is higher than that of mice given OP-PLA NPs. There was no difference between OP concentrations in other organs of OP-PEG-PLA group and OP-PLA group.

Figure 4. Tissue distribution of mice at 0.5 h after administration.

The mice given OP-PEG-PLA NPs had a significantly greater accumulative concentration of OP in the spleen than those administered with OP-PLA NPs (p < 0.05). Meanwhile, OP concentrations in the liver and the lung of OP-PEG-PLA group were remarkably smaller than those of OP-PLA NPs (p < 0.05) (see Figure 5).

Figure 5. Tissue distribution of mice at 4 h after administration.

At 4 h after administration, OP concentration in tumor increased in both NPs injected groups of mice compared to the OP solution injected ones (p < 0.05). Moreover, OP-PEG-PLA NPs had stronger tumor-targeting effect than OP-PLA NPs (see Figure 6).

Figure 6. Tissue distribution of mice at 12 h after administration.

Compared the three formats, the OP-PEG-PLA nanoparticle models had the biggest OP concentration in tumors at 12h after administration. Compared to OP solution models, both nanoparticle models had bigger concentrations in liver, spleen and lung (p < 0.05). OP-PEG-PLA NPs models showed a greater concentration in spleen and smaller in liver than OP-PLA NPs ones.

Discussions

Dialysis is employed to enrich NPs. The results showed that such particles were stable in PEG4000 and concentrating degree could be controlled (Vauthier et al. 2008). The concentration index (CI) of dialysis was 10. Particle size is one of the most important indicators which can evaluate the quality of concentrated NPs. NPs were concentrated considerably (CI≈11). Having measured the particle size of the concentrated NPs, we found that there was no obvious change. Consequently the particle size was not measured again after concentration. The dimethylformamide (DMF) in NPs mostly transferred to PEG4000 solution after concentration, and only about 0.1% DMF was left. Since the LD50 is 8.5 g/kg when DMF was given through skin, it proved that 0.1% DMF has a great safe range.

ICP-MS was used to detect the change of Pt concentration which can reflect the concentration of OP in plasma. ICP-MS applies to determination of various microelements, and it has a satisfactory good detection limit, wide linear range and flexible operating pattern. Moreover, ICP-MS has so good repeatability and accuracy that it is suitable for detection of OP. Pt does not change during the determination, so it is not necessary to investigate the influence of plasma.

Administration dosage was determined according to this formula: . According to administration principle of pharmacokinetics, the administration dosage means the treatment dosage. But the data in our research cannot reflect the change of OP in plasma concentration under treatment dosage. On the other hand, intravenous drip is the common method used in clinic instead of intravenous injection used in our research, so a lesser dose is a must for clinic for security.

We found that NPs prolonged the cycle time. We also found that OP-PEG-PLA NPs have the strongest effects. The hydrophobic shell on the surface of OP-PLA NPs promotes the uptake of those particles by mononuclear phagocytes. Since OP-PEG-PLA NPs have hydrophilic shell which hinders the phagocytosis of mononuclear phagocyte, then the OP-PEG-PLA NPs have a longer in vivo cycle time (Feng and Huang 2001, Schadlich et al. 2011).

NPs have sustained-release property which ensures that plasma drug concentration rises slowly and smoothly. For example, plasma OP concentrations in both nanoparticle groups at 0.5 h were lower than that of mice given OP solution. In other words, the elimination process of OP solution is, however, faster than that of NPs. The hydrophobic shell of OP-PLA NPs facilitates their enrichment in the spleen, lung and liver. Consequently, OP concentrations in organs of nanoparticle models were bigger than that of OP solution models.

Targeting effect of NPs is related to particle size, surface charge, surface modification and hydrophobic property (Brannon-Peppas and Blanchette 2004). Among them, particle size plays a significant role in penetrating bio-membrane. Passive targeting is available because tumor tissues have a high permeability (EPR effect). Since the hydrophobic shell on the surface of OP-PLA NPs boost the phagocytosis of the particles into mononuclear phagocytes, PEG is thus introduced to formulate a hydrophilic shell which resists plasma proteins and protects NPs from being degraded.

All results indicate that OP NPs improve drug distribution of tumor tissues. Moreover, both OP-PEG-PLA and PEG-PLA reduce the influences of OP on the brain and kidney, and raise the effects of OP on the liver, lung and spleen. Both formulations have no effect on the distribution of OP in the brain. Compared to OP-PLA NPs, OP-PEG-PLA NPs increase OP concentration in the tumor and spleen (Peracchia et al. 1999), improve the OP distribution in the spleen and reduce the OP distribution in the liver and lung. We concluded that PEG-modified NPs are suitable for medical treatment of tumor in organs except the liver and lung.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.

This work was supported by the National Natural Science Fund in 2014 (81373350) and Qingdao livelihood science & technology project fund in 2013 (13-1-3-49-nsh).