Cisplatin-loaded core cross-linked micelles: comparative pharmacokinetics, antitumor activity, and toxicity in mice

Figures & data

Table 1 Hemolytic activity of cisplatin/cl-micelles and empty cl-micelles against human red blood cells

Treatment	Concentration (μg/mL)	% hemolysis
Empty cl-micelles	40	0.00
	120	0.00
	400	0.08
Cisplatin/cl-micelles	60	0.00
	170	0.00
	500	0.02

Notes: Whole blood from a single healthy female volunteer was collected in tubes containing Li-heparin, centrifuged at 800 g for 10 minutes, rinsed two times with phosphate-buffered saline (PBS), and resuspended in PBS at 37°C. Micelles were then added to the blood cell suspension to obtain designated concentrations and a final erythrocyte concentration of 10⁸ cells/mL (PBS, pH 7.4). PBS and 1% Triton X-100 solutions added to the blood cells samples were used as negative and positive controls, respectively. The resulting suspensions were incubated at 37°C for 1 hour under agitation of 150 rpm, centrifuged at 800 × g (10 min) and analyzed for hemolysis in the supernatant using absorbance at 545 nm. The percentage of hemolysis was calculated as: Hemolysis (%) = (A_sample – A₀)/(A₁₀₀ – A₀) × 100%, where A_sample is the sample absorbance, A₀ and A₁₀₀ are absorbance values corresponding to PBS control and to the cells completely lysed with 1% Triton X-100. Data are mean from two different samples with an internal set of duplicates.

Table 2 Pharmacokinetic parameter estimates of platinum in plasma for cisplatin or cisplatin/cl-micelle-treated A2780 human ovarian cancer xenograft-bearing female nude micea

Parameter	Cisplatin	Cisplatin/cl-micelles
Cmaxb (μg/mL)	19.22	80.12
AUC (μg h/mL)	32.61	707.03
Cl (mL/h kg)	2.61	0.14
Vss (L/kg)	82.48	1.69
Λ	0.037	0.072
MRT (h)	31.63	12.37

Notes:

a The pharmacokinetic parameters were calculated using a noncompartment model with WinNonlin program.

b C_max represents the maximum observed concentration at the earliest evaluated time point of 5 min post dosage.

Abbreviations: C_max, maximum platinum concentration; AUC, area under the curve; Cl, clearance; V_ss, steady-state volume of distribution; Λ, apparent terminal elimination rate constant; MRT, mean residence time.

Figure 1 Concentration–time profile of platinum in A2780 human ovarian cancer xenograft-bearing female nude mice.

Notes: Platinum levels were measured following a single intravenous dose of cisplatin/cl-micelles (□) or cisplatin (■) at 7.5 mg cisplatin equivalents/kg body weight, in (A) plasma, (B) tumor, (C) kidney, (D) lung, (E) liver and (F) spleen. Values indicated are means ± standard error of the mean (n = 6), *P < 0.05.

Table 3 Pharmacokinetic parameter estimates in various tissues for cisplatin or cisplatin/cl-micelle-treated A2780 human ovarian cancer xenograft-bearing female nude mice

Parameter	Kidney		Spleen		Lung		Liver		Tumor
	Cis	Cis/cl-micelles	Cis	Cis/cl-micelles	Cis	Cis/cl-micelles	Cis	Cis/cl-micelles	Cis	Cis/cl-micelles
Tmax (h)	0.1	72.0	1.0	12.0	0.1	12.0	0.3	1.0	0.1	6.0
Cmax (μg/g tissue)	15.2	6.9	1.5	235.1	1.9	5.3	5.4	49.6	2.5	3.2
AUC (μg h/g tissue)	267	372	68	12115	59	284	235	2291	54	170

Abbreviations: Cis, cisplatin; T_max, time to reach C_max; C_max, maximum platinum concentration; AUC, area under the curve.

Figure 2 In vivo efficacy of cisplatin/cl-micelles in A2780 human ovarian cancer xenograft-bearing female nude mice.

Notes: Relative changes in (A) tumor volume and (B) body weight were measured following intravenous administration of cisplatin/cl-micelles (□) or cisplatin (■) at 4 mg cisplatin equivalents/kg body weight. Four administrations were given in total with each administration every fourth day. Control group received 100 μL of 5% dextrose (●). Values indicated are means ± standard error of the mean (n = 6). (C) Kaplan–Meier analysis of overall survival in: (1) control group, (2) cisplatin treatment group, and (3) cisplatin/cl-micelle treatment group.

Figure 3 Relative changes in body weight in C57Bl/6 mice.

Notes: (A) Cisplatin was injected intravenously every 4 days, four administrations in total, at a dose of 1 mg/kg (▲), 3 mg/kg (■), and 5 mg/kg (◆). (B) Cisplatin-loaded cl-micelles were injected at identical treatment schedule, at a cisplatin equivalent dose of 3 mg/kg (□), 5 mg/kg (⋄), and 10 mg/kg (○). Dextrose 5% treatment (●) was used as the control. Data are mean ± standard error of the mean (n = 3). Maximum tolerated dose was assumed to be 4 mg/kg in both treatment groups since the animals lost >20% body weight in both groups at the 5 mg/kg dose but did not lose any weight at the 3 mg/kg dose. Cisplatin-loaded cl-micelle treatment at 10 mg/kg resulted in toxic deaths in two animals. The remaining animals had >20% weight loss and were sacrificed by day 5.

Figure 4 Acute toxicity analysis in female C57Bl/6 mice.

Notes: (A) Changes in body weight (n = 5) until day 13 and day 28 were measured following intravenous administration of cisplatin/cl-micelles (□) or cisplatin (■) at 4 mg/kg. Vehicle control groups received empty micelles 2.5 mg/kg (△) or 100 μL of 5% dextrose (●). Four administrations were given in total with each administration at every fourth day. (B) Plasma BUN concentrations (n = 3–5) at the end of day 13 and day 28 of toxicity analysis. Treatment groups are: (1) control, (2) cisplatin treatment, (3) cisplatin/cl-micelle treatment, and (4) empty micelle treatment. Values indicated are means ± standard error of the mean.

Abbreviation: BUN, blood urea nitrogen.

Table 4 Clinical chemistry parameters from female C57Bl/6 mice at day 13 and day 28 of toxicity analysis

Treatment	Alk phos (U/L)	ALT (U/L)	AST (U/L)	BUN (mg/dL)	Creatinine (mg/dL)
Day 13
Control	117 ± 10	58 ± 17	69 ± 8	18 ± 2	a
Cisplatin	113 ± 16	a	115 ± 24b	75 ± 21b	a
Cisplatin/cl-micelles	111 ± 27	43 ± 9	81 ± 12c	22 ± 4c	a
Empty cl-micelles	128 ± 20	50 ± 9	59 ± 7c	18 ± 2c	a
Day 28
Control	136 ± 12	85 ± 28	d	20 ± 0	0.32 ± 0.05
Cisplatin	131 ± 12	112 ± 45	d	43 ± 6b	0.38 ± 0.03b
Cisplatin/cl-micelles	128 ± 25	93 ± 20	d	21 ± 5	0.29 ± 0.03
Empty cl-micelles	143 ± 15	100 ± 43	d	23 ± 5	0.34 ± 0.06

Notes: P values less than 0.05 were set as the level of significance. Data were expressed as mean ± standard deviation.

a These parameters could not be estimated due to inadequate blood draw.

b Represents significant difference from controls.

c Represents significant difference from free cisplatin.

d These parameters could not be accurately measured due to hemolysis in samples at blood draw.

Abbreviations: Alk phos, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; BUN, blood urea nitrogen.

Table 5 Tissue weights at sacrifice from female C57Bl/6 mice at day 13 and day 28 of toxicity analysis

Treatment	Liver/BW	Spleen/BW	Kidney/BW
Day 13
Control	0.0505 ± 0.0013	0.0040 ± 0.0006	0.0114 ± 0.0007
Cisplatin	0.0527 ± 0.0012	0.0028 ± 0.0003a	0.0145 ± 0.0010a
Cisplatin/cl-micelles	0.0500 ± 0.0030	0.0040 ± 0.0005	0.0116 ± 0.0005
Empty cl-micelles	0.0510 ± 0.0033	0.0040 ± 0.0009	0.0115 ± 0.0007
Day 28
Control	0.0471 ± 0.0041	0.0037 ± 0.0005	0.0117 ± 0.0006
Cisplatin	0.0542 ± 0.0025a	0.0040 ± 0.0007	0.0116 ± 0.0005
Cisplatin/cl-micelles	0.0461 ± 0.0048	0.0038 ± 0.0005	0.0119 ± 0.0007
Empty cl-micelles	0.0468 ± 0.0018	0.0036 ± 0.0004	0.0120 ± 0.0008

Notes:

a Represents significant difference from controls. P values less than 0.05 were set as the level of significance. Data were expressed as mean ± standard deviation.

Abbreviation: BW, body weight.

Figure 5 Histopathological changes in kidney and bone marrow tissues from toxicity analysis in female C57Bl/6 mice.

Notes: Light microscopy images of hematoxylin and eosin-stained kidney (A–D, day 13; I–L, day 28) and bone marrow (E–H, day 13; M–P, day 28) were taken at a magnification of 200× and 100× respectively. Tissue samples were collected at day 13 and day 28 following intravenous administration of cisplatin/cl-micelles or cisplatin at 4 mg/kg body weight. Vehicle control groups received 2.5 mg free micelles/kg body weight or 100 μL of 5% dextrose. Four administrations were given in total, each administration performed every fourth day.

Figure 6 Light microscopy images (original magnification 100×) of hematoxylin and eosin-stained liver and spleen tissues from toxicity analysis in female C57Bl/6 mice.

Notes: No histopathological changes were observed in liver (A and B, day 13; E and F, day 28) and spleen (C and D, day 13; G and H, day 28) following administration of free cisplatin at a dose of 4 mg/kg, cisplatin-loaded cl-micelles at a cisplatin equivalent dose of 4 mg/kg, or empty micelles at 2.5 mg polymer/kg body weight. Treatment was administered by bolus intravenous tail vein injection (n = 5), four treatments in total, with each treatment at a 4-day interval. The animals were sacrificed using CO₂ euthanasia on day 13 and day 28 for toxicity analysis. Hematoxylin and eosin staining was performed as described in materials and methods.

Figure 7 Immunohistochemistry images of PEG stained tissues from acute toxicity analysis in female C57Bl/6 mice.

Notes: No accumulation was observed in the kidney (magnification 200×) from either cisplatin-loaded (A, cortex; B, tubules) or empty micelles (C, cortex; D, tubules) treatment groups. Liver (E–F, cisplatin/cl-micelles; G–H, empty micelle) and spleen (I–J, cisplatin/cl-micelles; K–L, empty micelle) showed strong accumulation of micelles (magnification 200×/400×). Spleen shows accumulation of micelles only in the red pulp region.

Figure 8 Uptake of cisplatin/cl-micelles by macrophages.

Notes: Female nude mice (n = 3) bearing A2780 ovarian cancer xenografts (approximately 800 mm³), administered a single intravenous tail vein injection of free cisplatin or cisplatin/cl-micelles (7.5 mg/kg), or empty micelles (20 mg polymer/kg body weight) were sacrificed 24 hours post-treatment and liver, spleen, and tumors were harvested for flow cytometric analysis. Staining was performed with anti-CD14 antibody and anti- PEG antibody as described in material and methods. (A) Relative population of CD14 positive cells amongst the treatment groups was similar. (B) and (C), majority of the PEG positive cells in the micelle treatment group liver and spleen costained for mouse CD14 (70%–80%), indicating macrophage uptake of micelles in these organs. Tumor had two distinct populations of cells with about 30%–40% of PEG positive cells costaining for CD14 while the remaining associated with other tumor cells.

Figure 9 Immunofluorescence images (original magnification 200×) of liver and spleen tissues from toxicity analysis in female C57Bl/6 mice.

Notes: Slide mounted sections (5 μm) of liver (A, day13; B, day 28) and spleen (C, day 13; D, day 28) following administration of cisplatin-loaded cl-micelles at a cisplatin equivalent dose of 4 mg/kg were stained for PEG (anti-PEG antibody, green) and nuclei (4′,6-diamidino-2-phenylindole, blue). Significant decrease in PEG staining in day 28 tissues was seen relative to corresponding day 13 tissues. Treatment was administered by bolus intravenous tail vein injection (n = 5), four treatments in total, with each treatment at a 4-day interval. The animals were sacrificed using CO₂ euthanasia on day 13 and day 28 for toxicity analysis.

Abbreviation: PEG, poly(ethylene glycol).