Dual targeting curcumin loaded alendronate-hyaluronan- octadecanoic acid micelles for improving osteosarcoma therapy

Figures & data

Figure 1 Schematic representation for the synthesis of (A) hyaluronic acid-octadecanoic acid (HA-C₁₈) and (B) alendronate-hyaluronic acid-octadecanoic acid (ALN-HA-C₁₈).

Table 1 The orthogonal design factors and levels

Independent variables	Levels
	1	2	3
X1: The mass ratio of polymer to drug	5: 1	10: 1	15: 1
X2: Type of organic solvent	Ethanol	Acetone	Chloroform
X3: Solidification temperature (°C)	40	60	80

Figure 2 ¹H NMR spectrum of (A) hyaluronic acid (HA) and (B) alendronate-hyaluronic acid-octadecanoic acid (ALN-HA-C₁₈).

Figure 3 Plots of I₁/I₃ against the logarithim of the ALN-HA-C₁₈ concentration.

Table 2 The orthogonal design L9 (3⁴) and resulting values

Experiments	Variables			Responses
X1: Polymer to drug	X2: Type of organic solvent	X3: Solidification temperature (°C)	Y1: Particle size (nm)	Y2: EE% (%)
1	1	1	1	244.4	12.3
2	1	2	2	248.5	37.6
3	1	3	3	324.2	9.7
4	2	1	2	122.2	36.6
5	2	2	3	115.3	34.4
6	2	3	1	472.5	8.2
7	3	1	3	200.8	18.0
8	3	2	1	220.2	32.3
9	3	3	2	377	10.8
K1	272.4	189.1	312.4
K2	236.7	188.2	249.2
K3	266	391.2	213.4
R	35.7	202.1	99
K1’	19.9	22.3	17.6
K2’	26.4	34.8	28.3
K3’	20.4	16.7	20.7
R’	6.5	18.1	10.7

Figure 4 The size distribution (A) and transmission electron microscopy (TEM) image (B) of CUR loaded ALN-HA-C₁₈ micelles. (C) In vitro release characteristics of free CUR and CUR loaded ALN-HA-C₁₈ micelles in 1% Tween 80 PBS medium. (D) Binding ratio of CUR, CUR loaded HA-C₁₈ micelles, and CUR loaded ALN-HA-C₁₈ micelles with hydroxyapatite. Data are presented as the mean ± SD (n=3). ***Significant difference between the two groups (***P＜0.001).

Figure 5 In vitro cytotoxicity of blank ALN-HA-C₁₈ micelles, free CUR, and CUR loaded ALN-HA-C₁₈ micelles against HOB cells (A) and MG-63 osteosarcoma cells (B). Fluorescence microscope images (C) and flow cytometry analysis (D) of MG-63 cells incubated with free C6, C6 loaded ALN-HA-C₁₈ micelles with HA and C6 loaded ALN-HA-C₁₈ micelles. Data are presented as the mean ± SD (n=6). *, ** and *** represent P＜0.05, P＜0.01 and P＜0.001, respectively. The bar is 50 μm.

Figure 6 The in vivo antitumor activity of CUR loaded ALN-HA-C₁₈ micelles on osteosarcoma bearing nude mice. (A) Tumor volume changes; and (B) body weight changes. (C) The hematoxylin and eosin (H&E) staining of the heart, liver, spleen, lung and kidney in saline and CUR loaded ALN-HA-C₁₈ micelles group after treatment. Data are presented as the mean ± SD (n=6). *Represents P＜0.05. Bars =100 μm.