中国药学杂志

Independent variables	Coded levels
X₁:PVP K30/g	1	2	3
X₂:CS/g	1	2	3
X₃:Liquid/%	45	55	65

Independent variables	Coded levels
X₁:PVP K30/g	1	2	3
X₂:CS/g	1	2	3
X₃:Liquid/%	45	55	65

Project	Standard for evaluation
Microneedle body morphology	Severe bending or majority fracture (0-3 points). Bending or a few breaks (4-10 points). Slightly curved, does not affect the use (11-13 points). No bending and no breaking (14-15 min)
Film-formation	Can not form film, after drying in the mold is broken slag (0-2 points). After drying and curing, it becomes film-like, has severe shrinkage or is easy to break (3-5 minutes). Slightly crumpled or cracked after drying and curing, but does not affect the use (6-10 points). After drying and curing, a complete array of microneedles of moderate thickness is formed (11-15 min)
Tip hardness	Can not Pierce aluminum foil (0-1 score). It could puncture aluminum foil but could not puncture rat skin, and the tip of the needle broke (2-5 points). Can puncture aluminum foil and rat skin without breaking the tip (6-10 minutes)
Backing performance	The backing is obviously crumpled, and the pressure is fragile (0-2 points). The backing has no obvious shrinkage, but presents a curved bulge, and is fragile (3-6 points) under excessive pressure. Flat backing without wrinkling, pressure is not easy to break (7-10 points)
Number of bubbles	Filled with a large number of bubbles or forming many empty needles, so that microneedles cannot be used (0-1 score). There are a few bubbles or slightly empty needles (2-5 points). There is a small amount of bubbles but it does not affect the use (6-9 points). No visible bubbles (10 points)
Microneedle body integrity rate	A large number of needle bodies are missing, making microneedles unusable (0-1 score). Half of the needle body is missing (2-3 points). A small amount of needle body missing, does not affect the use of (4-9 points). Absence of needle body (10 points)

Project	Standard for evaluation
Microneedle body morphology	Severe bending or majority fracture (0-3 points). Bending or a few breaks (4-10 points). Slightly curved, does not affect the use (11-13 points). No bending and no breaking (14-15 min)
Film-formation	Can not form film, after drying in the mold is broken slag (0-2 points). After drying and curing, it becomes film-like, has severe shrinkage or is easy to break (3-5 minutes). Slightly crumpled or cracked after drying and curing, but does not affect the use (6-10 points). After drying and curing, a complete array of microneedles of moderate thickness is formed (11-15 min)
Tip hardness	Can not Pierce aluminum foil (0-1 score). It could puncture aluminum foil but could not puncture rat skin, and the tip of the needle broke (2-5 points). Can puncture aluminum foil and rat skin without breaking the tip (6-10 minutes)
Backing performance	The backing is obviously crumpled, and the pressure is fragile (0-2 points). The backing has no obvious shrinkage, but presents a curved bulge, and is fragile (3-6 points) under excessive pressure. Flat backing without wrinkling, pressure is not easy to break (7-10 points)
Number of bubbles	Filled with a large number of bubbles or forming many empty needles, so that microneedles cannot be used (0-1 score). There are a few bubbles or slightly empty needles (2-5 points). There is a small amount of bubbles but it does not affect the use (6-9 points). No visible bubbles (10 points)
Microneedle body integrity rate	A large number of needle bodies are missing, making microneedles unusable (0-1 score). Half of the needle body is missing (2-3 points). A small amount of needle body missing, does not affect the use of (4-9 points). Absence of needle body (10 points)

Run	Variables	Responses
1	1	2	65	13	8	9	8	6	10	54
2	3	2	55	13	12	7	7	6	6	51
3	3	3	65	14	10	8	9	5	6	52
4	3	1	55	13	12	7	8	5	4	49
5	2	3	55	10	7	8	4	7	6	42
6	1	3	45	13	11	6	8	7	9	54
7	3	1	65	12	12	8	8	6	9	55
8	1	1	45	14	10	8	9	9	10	60
9	1	3	65	13	8	10	8	8	8	55
10	2	1	45	13	12	8	7	10	7	57
11	2	2	55	10	14	4	8	10	10	56
12	2	3	65	12	7	6	5	9	9	48
13	1	2	55	12	14	5	8	9	10	58
14	3	2	65	14	13	8	8	10	10	63
15	2	1	65	13	8	8	6	10	9	54
16	1	3	55	12	6	4	6	8	9	45
17	3	1	45	8	11	6	7	6	10	48
18	2	3	45	12	12	1	6	9	10	50
19	3	2	45	10	7	3	5	5	9	39
20	1	2	45	10	12	4	8	9	9	52
21	2	1	55	10	12	7	9	8	9	55

Run	Variables	Responses
1	1	2	65	13	8	9	8	6	10	54
2	3	2	55	13	12	7	7	6	6	51
3	3	3	65	14	10	8	9	5	6	52
4	3	1	55	13	12	7	8	5	4	49
5	2	3	55	10	7	8	4	7	6	42
6	1	3	45	13	11	6	8	7	9	54
7	3	1	65	12	12	8	8	6	9	55
8	1	1	45	14	10	8	9	9	10	60
9	1	3	65	13	8	10	8	8	8	55
10	2	1	45	13	12	8	7	10	7	57
11	2	2	55	10	14	4	8	10	10	56
12	2	3	65	12	7	6	5	9	9	48
13	1	2	55	12	14	5	8	9	10	58
14	3	2	65	14	13	8	8	10	10	63
15	2	1	65	13	8	8	6	10	9	54
16	1	3	55	12	6	4	6	8	9	45
17	3	1	45	8	11	6	7	6	10	48
18	2	3	45	12	12	1	6	9	10	50
19	3	2	45	10	7	3	5	5	9	39
20	1	2	45	10	12	4	8	9	9	52
21	2	1	55	10	12	7	9	8	9	55

Model	SD	CV/%	r²	Adjusted R²	Predicted R²	PRESS	Result
Linear	5.14	9.83	0.324 5	0.205 3	-0.010 5	670.80
2FI	4.46	8.54	0.580 6	0.400 9	0.042 2	635.76
Quadratic	0.969 0	1.85	0.984 4	0.971 7	0.932 6	44.76	Suggested
Cubic	0.802 8	1.54	0.995 1	0.980 6	0.858 8	93.76	Aliased

Model	SD	CV/%	r²	Adjusted R²	Predicted R²	PRESS	Result
Linear	5.14	9.83	0.324 5	0.205 3	-0.010 5	670.80
2FI	4.46	8.54	0.580 6	0.400 9	0.042 2	635.76
Quadratic	0.969 0	1.85	0.984 4	0.971 7	0.932 6	44.76	Suggested
Cubic	0.802 8	1.54	0.995 1	0.980 6	0.858 8	93.76	Aliased

Source	df	Coefficient estimate	Sum of squares	Mean square	F	P
Model	9		653.48	72.61	77.33	<0.000 1
Intercept	1	60.44
A-PVP K30	1	3.26	131.32	131.32	139.86	<0.000 1
B-CS	1	-1.24	19.1	19.1	20.34	0.000 9
C-Liquid	1	-1.29	21.89	21.89	23.32	0.000 5
AB	1	4.06	59.21	59.21	63.06	<0.000 1
AC	1	1.11	7.84	7.84	8.35	0.014 7
BC	1	-0.222 2	0.313 7	0.313 7	0.334 1	0.574 9
A²	1	-2.00	11.37	11.37	12.11	0.005 2
B²	1	-2.50	17.76	17.76	18.92	0.001 2
C²	1	-6.78	190.82	190.82	203.23	<0.000 1
Residual	11		10.33	0.938 9
Cor Total	20		663.81

Source	df	Coefficient estimate	Sum of squares	Mean square	F	P
Model	9		653.48	72.61	77.33	<0.000 1
Intercept	1	60.44
A-PVP K30	1	3.26	131.32	131.32	139.86	<0.000 1
B-CS	1	-1.24	19.1	19.1	20.34	0.000 9
C-Liquid	1	-1.29	21.89	21.89	23.32	0.000 5
AB	1	4.06	59.21	59.21	63.06	<0.000 1
AC	1	1.11	7.84	7.84	8.35	0.014 7
BC	1	-0.222 2	0.313 7	0.313 7	0.334 1	0.574 9
A²	1	-2.00	11.37	11.37	12.11	0.005 2
B²	1	-2.50	17.76	17.76	18.92	0.001 2
C²	1	-6.78	190.82	190.82	203.23	<0.000 1
Residual	11		10.33	0.938 9
Cor Total	20		663.81

Group	Model	Equation	Parameter	r²
High-dose	zero-order	F=k₀·t	k₀=9.670	0.482 3
	first-order	F=M·[1-Exp(-k₁·t)]	k₁=0.418, M=322.22	0.965 9
	Higuchi	F=k_H·t^0.5	k_H=64.555	0.680 8
Low-dose	zero-order	F=k₀·t	k₀=6.751	0.819 3
	first-order	F=M·[1-Exp(-k₁·t)]	k₁=0.139, M=169.36	0.991 2
	Higuchi	F=k_H·t^0.5	k_H=40.812	0.948 2

Group	Model	Equation	Parameter	r²
High-dose	zero-order	F=k₀·t	k₀=9.670	0.482 3
	first-order	F=M·[1-Exp(-k₁·t)]	k₁=0.418, M=322.22	0.965 9
	Higuchi	F=k_H·t^0.5	k_H=64.555	0.680 8
Low-dose	zero-order	F=k₀·t	k₀=6.751	0.819 3
	first-order	F=M·[1-Exp(-k₁·t)]	k₁=0.139, M=169.36	0.991 2
	Higuchi	F=k_H·t^0.5	k_H=40.812	0.948 2

Group	Ca²⁺	P³⁺	ALP
Sham	2.291±0.042²⁾	1.565±0.154¹⁾	20.596±8.549²⁾
Control	2.063±0.037⁴⁾	1.411±0.159³⁾	42.393±13.387⁴⁾
DMN-Blank	2.045±0.037⁴⁾	1.555±0.075	38.922±6.992⁴⁾
DMN-Low	2.067±0.056⁴⁾	1.535±0.063	30.610±4.701¹⁾
DMN-High	2.140±0.037¹⁾⁴⁾	1.408±0.082³⁾	23.526±4.904²⁾
Gavage	2.173±0.069²⁾⁴⁾	1.496±0.107	27.138±6.039²⁾

Group	Ca²⁺	P³⁺	ALP
Sham	2.291±0.042²⁾	1.565±0.154¹⁾	20.596±8.549²⁾
Control	2.063±0.037⁴⁾	1.411±0.159³⁾	42.393±13.387⁴⁾
DMN-Blank	2.045±0.037⁴⁾	1.555±0.075	38.922±6.992⁴⁾
DMN-Low	2.067±0.056⁴⁾	1.535±0.063	30.610±4.701¹⁾
DMN-High	2.140±0.037¹⁾⁴⁾	1.408±0.082³⁾	23.526±4.904²⁾
Gavage	2.173±0.069²⁾⁴⁾	1.496±0.107	27.138±6.039²⁾

利塞膦酸钠可溶性微针的制备及预防绝经后骨质疏松症研究

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梁俊晖 ¹ , 苏安宇 ¹ , 杨雅雯 ² , 李亚欣 ¹ , 边影彤 ¹ , 邓世森 ¹ , 袁秋虹 ¹ , 王利胜 ¹^,^*

中国药学杂志 | 论著 2024,59(16): 1522-1531

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中国药学杂志 | 论著 2024, 59(16): 1522-1531

利塞膦酸钠可溶性微针的制备及预防绝经后骨质疏松症研究

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梁俊晖¹, 苏安宇¹, 杨雅雯², 李亚欣¹, 边影彤¹, 邓世森¹, 袁秋虹¹, 王利胜¹^,^*

作者信息

¹ 广州中医药大学中药学院, 广州 510006

² 帝国理工学院生命科学与医学学院,伦敦 SW72AZ

梁俊晖,男,硕士研究生研究方向:药物制剂新技术与新剂型

通讯作者:

* 王利胜,男,博士,教授,博士生导师研究方向:药物制剂新技术与新剂型 Tel:(020)39358290

Fabrication of Risedronate Sodium-Loaded Dissolving Microneedles and Evaluation of Its Therapeutic Effects on Postmenopausal Osteoporosis

Junhui LIANG¹, Anyu SU¹, Yawen YANG², Yaxin LI¹, Yingtong BIAN¹, Shisen DENG¹, Qiuhong YUAN¹, Lisheng WANG¹^,^*

Affiliations

¹ School of Chinese Materia Medica, Guangzhou University of Chinese Medicine, Guangzhou 510006, China

² Faculty of Life Sciences & Medicine, Imperial College London, London SW72AZ, United Kingdom

出版时间: 2024-08-22 doi: 10.11669/cpj.2024.16.010

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摘要

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目的制备利塞膦酸钠可溶性微针,对其进行表征及预防绝经后骨质疏松症药效评价。方法通过响应面法优选微针的制备工艺;采用扫描电镜、皮内溶解试验、穿刺试验、皮肤屏障恢复试验与体外透皮试验对所制得微针的外观、溶解性能、机械性能、安全性、透皮特性进行表征;采用卵巢切除骨质疏松症模型大鼠进行药效学评价。结果经过优选的微针配方为聚乙烯吡咯烷酮-硫酸软骨素(PVP K30-CS)配比为1:0.86,溶剂占比55%;所得微针具有良好的性状,并展现出与口服给药相似的Ca²⁺、P³⁺和碱性磷酸酶(alkaline phosphatase,ALP)水平调节能力,显著提升模型大鼠骨密度并修复其骨微结构。结论利塞膦酸钠可溶性微针质量稳定、使用方便、药效确切,是干预绝经后骨质疏松症的潜在选项。

关键词

利塞膦酸钠 / 双膦酸盐 / 可溶性微针 / 骨质疏松 / 响应面法

Abstract

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OBJECTIVE To prepare risedronate sodium (RIS)-loaded dissolving microneedle (DMN) and evaluate its efficacy in preventing postmenopausal osteoporosis. METHODS The preparation process of RIS-DMN was optimized by Box-Behnken design of response surface methodology. The appearance, solubility, mechanical property, safety and transdermal effect of RIS-DMN were characterized by scanning electron microscope, intradermal dissolution test, puncture test, skin barrier recovery test and in vitro permeation test. The pharmacodynamic evaluation of RIS-DMN was performed in ovariectomized osteoporosis model rats. RESULTS The optimal formulation were determined to be 45% for solute (mixed with 1:0.86 PVP K30 and CS) and 55% for solvent. It was found that the RIS-DMN have good physical characteristics and properties, and showed great effects in regulating the level of Ca²⁺, P³⁺and alkaline phosphatase (ALP). Meanwhile, the RIS-DMN showed great effects in repairing bone microstructure and improving bone density in ovariectomized osteoporosis model rats, as similar as oral administration. CONCLUSION The RIS-DMN has stable quality, convenient use and precise efficacy, shows great potential in the treatment of postmenopausal osteoporosis.

Key words

risedronate sodium / bisphosphonate / dissolving microneedle / osteoporosis / response surface

引用本文

梁俊晖, 苏安宇, 杨雅雯, 李亚欣, 边影彤, 邓世森, 袁秋虹, 王利胜. 利塞膦酸钠可溶性微针的制备及预防绝经后骨质疏松症研究. 中国药学杂志, 2024 , 59 (16) : 1522 -1531 . DOI: 10.11669/cpj.2024.16.010

Junhui LIANG, Anyu SU, Yawen YANG, Yaxin LI, Yingtong BIAN, Shisen DENG, Qiuhong YUAN, Lisheng WANG. Fabrication of Risedronate Sodium-Loaded Dissolving Microneedles and Evaluation of Its Therapeutic Effects on Postmenopausal Osteoporosis[J]. Chinese Pharmaceutical Journal, 2024 , 59 (16) : 1522 -1531 . DOI: 10.11669/cpj.2024.16.010

正文

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骨质疏松症是人类最常见的骨病,尤见于绝经后妇女。骨密度降低、骨微结构破坏和骨脆性增加是骨质疏松症的主要特征,不仅给患者带来巨大的骨折风险,同时极易引发多种并发症^[1-2]。相关研究显示,我国40岁以上绝经后女性骨质疏松症患病率高达32.5%,是相同年龄段男性的4~5倍,显著高于欧美国家^[3-5]。同时,由于骨质疏松症早期缺乏典型症状,患者通常在发生骨折等不可逆伤害后才得以确诊^[6]。因此针对骨质疏松症好发人群尽早采取预防治疗意义重大。

双膦酸盐是目前临床上应用最为广泛的抗骨质疏松药物^[7]。美国临床内分泌学家协会和美国内分泌学学会《绝经后女性骨质疏松诊断与治疗临床实践指南(2020)》^[8]指出,对于大部分高风险骨折患者而言,可首选双膦酸盐进行初始治疗。利塞膦酸钠(risedronate sodium,RIS)作为双膦酸盐类的第三代药物,具有强大的抗骨吸收作用及较广抗骨折谱,是目前最重要的一类抗骨吸收抑制剂,尤其适用于绝经后骨质疏松症的防治^[9-10]。双膦酸盐的不良反应主要为消化道损伤、肾毒性及眼损伤,这与其口服及静脉注射的给药方式息息相关^[11-12]。此外口服方式要求患者服药后半小时内保持坐姿或直立姿势以减轻双膦酸盐对消化道的损伤^[13],这对于骨质疏松症患者尤其是重症患者而言,无疑是十分困难的。

经皮给药系统(transdermal drug delivery system, TDDs)是继口服、注射之后的第三大给药系统,突破了传统给药方式首过代谢、峰谷现象、依从性差等局限^[14-15]。利用TDDs进行双膦酸盐给药能够很好地解决其口服及注射带来的不良反应,然而相关研究甚少^[16]。双膦酸盐的高亲水性与相对较大的相对分子质量是阻碍自身经皮吸收的主要因素^[17-18]。随着学科的日益发展,微针技术为双膦酸盐的经皮递送带来了可能。微针是一种微型矩阵针体给药技术,能够定向突破皮肤屏障,使亲水性小分子药物、生物大分子药物等原本不适于经皮给药的药物直接进入真皮层,是将大分子和亲水性药物经皮肤输送到全身循环的有效方法,近年来已成为TDDs的热点研究方向^[19-20]。其中,可溶性微针(dissolving microneedle,DMN)以其制备过程简单、成本低、载药量大以及不产生针尖废弃物等优点而备受关注^[21]。

本研究的目的是开发一种以RIS为模型药的DMN,并对其在卵巢去势大鼠骨质疏松症的预防作用进行评价,为双膦酸盐的临床经皮应用提供理论依据。

1 实验材料

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1.1 试剂与仪器

聚二甲基硅氧烷(PDMS),雌二醇(E2)放射免疫测定试剂盒(武汉优尔生科技股份有限公司);血清碱性磷酸酶(ALP)测定试剂盒(深圳雷杜生命科学股份有限公司);磷(P³⁺)测定试剂盒、钙(Ca²⁺)测定试剂盒(长春汇力生物技术有限公司);RIS(批号Y17N8C48386,纯度98%,上海源叶生物科技有限公司);聚乙烯吡咯烷酮(PVP K30,上海麦克林生化科技有限公司);硫酸软骨素(CS,西安西海生物科技有限公司);甲醇为色谱纯;实验用水均为超纯水,其他试剂均为分析纯。

酶标检测仪(美国BioTeK公司);全自动生化分析仪(深圳雷杜生命科学股份有限公司);双能X线骨密度检测仪(美国GE公司,型号:Prodigy);干加热自动透皮系统(美国LOGAN公司,型号:SYSTEM 918-12);高效液相色谱仪(悟空仪器公司,型号:K2025);十万分之一电子分析天平(日本岛津公司,型号:AUW120D);高速组织研磨仪(上海净信实业发展有限公司,型号:KZ-Ⅱ);台式高速冷冻离心机(北京大龙兴创实验仪器有限公司,型号:D1524R);超纯水机(上海力辰邦西仪器科技有限公司,型号:UPTA-20);微针模具(15 mm×15 mm,单针高度和直径分别为900和450 μm,台州薇凯生物科技有限公司)。

1.2 实验动物

SPF级雌性Wistar大鼠,体重230~250 g,由广州中医药大学实验动物中心提供,许可证号:SYXK(粤)2019-0202。动物饲养于SPF级环境中,温度20~25℃,湿度50%~60%,白昼光照周期12 h/12 h,自由饮水和进食。适应性饲养1周后进行实验。本研究中所有涉及动物的实验均得到实验动物伦理委员会的许可,并严格遵循实验动物福利伦理原则执行,伦理审查编号:ZYD-2022-021。

2 实验方法

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2.1 模具的制备

称取等量聚二甲基硅氧烷(PDMS)A液与B液于烧杯中,持续搅拌10 min使充分混匀。将混合液于4 ℃环境下以3 000 r·min^-1的转速离心10 min,随后倒入金属阳模中进行真空脱气。脱气完毕后,在60 ℃环境下固化90 min,随后冷却至室温。将PDMS阴模从金属阳模中剥离,即得PDMS微针模具。

2.2 微针的制备

将单一或复合基质材料充分溶胀于浓度为10 mg·mL^-1的利塞膦酸钠水溶液中并搅拌均匀,注入“2.1”项所制得PDMS微针模具中。在4 ℃条件下,将注入基质液的PDMS模具以3 000 r·min^-1的转速离心20 min。使用平整的玻璃板小心刮去模具表面多余的基质液,随后放入干燥器中室温干燥48 h取出,小心脱模,即得微针。

2.3 微针配方的优选

前期及相关研究表明^[22-24], PVP K30、CS以及含有 CS的复合材料所制备的微针具有较好的机械强度及韧性,其中以PVP K30-CS(质量比1:1)制备出来的微针容易揭膜、阵列完整、硬度最好,满足本研究对微针性能的需求,故选择PVP K30与CS作为利塞膦酸钠可溶性微针(RIS-DMN)的基质材料。

在此基础上,以PVP K30、CS、溶剂含量作为关键自变量因素建立三因素、三水平的Box-Behnken设计,对制得微针成型性各项指标进行量化分级与加权评分,通过Design-Expert 12.0软件进行响应面优化研究,变量设计见表1。微针成型性评分标准见表2。

2.4 微针表征

根据“2.3”项优选的制备工艺,参照“2.2”项下方法制备RIS-DMN并对其进行表征。

表观形貌:使用肉眼与扫描电镜观察可溶性微针的表观形貌特征。

微针皮内溶解实验:将微针垂直刺入健康Wistar大鼠去毛腹部皮肤,按压1 min。停止按压后,分别于5、10、20、30 min取下微针,在光学显微镜下观察针体溶解状况。

机械性能考察:取健康Wistar大鼠,采用直接脱颈椎法实施安乐死。使用动物理发器小心清除大鼠腹部毛发并取下大鼠腹部皮肤,用生理盐水冲洗干净,滤纸吸干。分别采用铝箔穿刺试验和离体大鼠皮肤穿刺试验对微针的机械性能进行考察。铝箔穿刺时,将微针垂直刺入铝箔中,按压1 min;离体大鼠皮肤穿刺时,以同样方法将微针垂直刺入健康无损伤大鼠离体皮肤,按压1 min后取出微针,立即用质量分数0.4%台盼蓝溶液对皮肤针孔进行染色,15 min后用蘸有异丙醇的棉球擦除大鼠皮肤表面多余的染色剂,并使用蘸有生理盐水的棉球进行再次擦洗,随后观察皮肤表面染色小孔状况。

皮肤屏障恢复实验:以质量分数0.4%台盼蓝水溶液作为溶剂,按“2.2”项下方法制备DMN。将台盼蓝水溶液作为溶剂的DMN垂直刺入大鼠去毛腹部皮肤,按压1 min后取下。观察取下微针后0、5、30、120、240、360、480 min后皮肤中台盼蓝的扩散情况,以此判断微针对大鼠皮肤角质层的破坏作用。

2.5 微针释放特性评价

色谱条件:RIS的高效液相色谱检测条件:色谱柱:ChromCore C₁₈ (4.6 mm×250 mm,5 μm),流动相:磷酸二氢钠缓冲液(含5 mmol·L^-1磷酸二氢钠,2 mmol·L^-1四丁基溴化铵,1.5 mmol·L^-1乙二胺四乙酸二钠,用质量分数4%氢氧化钠溶液调节pH至7.2) -甲醇=75:25,流速:1.0 mL·min^-1,检测波长:262 nm,柱温:30 ℃,进样量:20 μL。RIS的保留时间约为7.8 min,总运行时间为10 min。

针头载药量测定:按“2.2”项下方法,分别以10 mg·mL^-1与5 mg·mL^-1的RIS水溶液制备高、低剂量RIS-DMN。使用手术刀分别将2种DMN针头部分完全分离出来,加入10 mL流动相,室温下超声60 min以充分溶解,随后通过0.22 μm微孔滤膜,用HPLC法测定2种微针的RIS含量。

体外透皮实验:取健康Wistar大鼠,采用直接脱颈椎法实施安乐死。使用动物理发器小心清除大鼠腹部毛发并取下大鼠腹部皮肤,剔除皮下脂肪组织,用生理盐水冲洗干净,滤纸吸干。将制得的高、低剂量RIS-DMN分别垂直刺入处理好的离体鼠皮上,按压1 min,随后使用医用胶带固定,安装于改良Franz扩散池的给药口和接收池之间,表皮层朝向给药口。扩散池的扩散面积为1.77 cm²,接收池体积为5 mL。以生理盐水作为接收液,温度保持在(32±0.5)℃,转速100 r·min^-1。分别于透皮0.5、1、2、3、4、6、8、12、16、24 h时间点收集2 mL接收液作为样品进行含量测定,取样后接收池及时补充等量生理盐水。将取出的接收液样品通过0.22 μm微孔滤膜,随后用HPLC法进行含量测定,并按公式1计算高剂量RIS-DMN在每个时间点的累积透过量:

公式(1)Q_n=

ρ n V + ∑ i = 1 n - 1 ρ i V i S

其中,Q_n为累积透过量,ρ_n为第n次取样时测得的药物质量浓度(μg·mL^-1),V为接收池内接收液的总体积(V=5 mL),ρ_i为第i次取样时测得的药物质量浓度(μg·mL^-1),V_i为取样体积(V_i=2 mL),i=n-1,S为扩散渗透面积(S=1.77 cm²)。

2.6 药效学研究

动物造模、分组与给药: 本实验采用双侧卵巢摘除术建立绝经后骨质疏松症大鼠模型。取健康雌性Wistar大鼠30只,随机分为6组:对照组、假手术组、灌胃组(按体质量0.7 mg·kg^-1给予RIS,生理盐水配制)、空白微针组、低剂量组(5 mg·mL^-1 RIS水溶液制备的RIS-DMN)、高剂量组(使用10 mg·mL^-1 RIS水溶液制备的RIS-DMN)。手术后1周开始给药,每周1次,连续3个月。假手术组和对照组使用灌胃组等量生理盐水灌胃;微针组每周使用1片相应的微针。

血清生化指标测定:给药3个月后,使用质量分数3%戊巴比妥注射麻醉大鼠,进行腹主动脉取血。离心取血清放入-80 ℃保存。使用全自动生化仪测定血清中的ALP、Ca²⁺、P³⁺水平。

骨密度测定:取血后采用直接脱颈椎法实施安乐死,取大鼠左后肢股骨,剔除附着的软组织与肌肉,使用双能X线骨密度测量仪测定骨密度。

组织病理学检查:取大鼠左后肢胫骨于体积分数4%多聚甲醛中固定2 d,用牙科电锯从矢状面剖开,暴露骨髓腔并取左胫骨近端制备常规脱钙骨组织蜡块,切4 μm厚度骨组织切片,HE染色,在光学显微镜下观察骨组织形态变化。

数据处理: 采用SPSS Statisitics 20软件对所采集的数据进行统计学分析,计量资料符合正态分布的采用($\bar{x}±s$)进行描述。不符合正态分布时,采用非参数检验。符合正态分布的数据进行方差齐性检验后,采用单因素方差分析,多组间两两比较采用最小显著差异(LSD)法。当P<0.05时,认为有统计学差异。

3 实验结果

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3.1 微针配方的优选

表3为15个组别制得微针(n=3)的成型性综合评分。表4显示响应面的不同模型拟合情况,其中,较高的r²与最小的PRESS值显示实验数据与二次模型具备最佳的拟合性。

模型参数的回归系数和方差分析(ANOVA)见表5。PVP K30的加入量(X₁)、CS的加入量(X₂)、溶剂在微针中的占比(X₃)均对RIS-DMN成形性的影响显著;而在交叉乘积项上PVP K30加入量的平方(

X 1 2

)、CS加入量的平方(

X 2 2

)、溶剂占比的平方(

X 3 2

)对微针成形性的影响显著;交互项上X₁X₂、X₁X₃对微针成形性有影响显著。各因素经回归拟合后,得公式2。

公式(2)Y=60.44+3.26X₁-1.24X₂-1.29X₃+4.06X₁X₂+1.11X₁X₃-0.222 2X₂X₃-2.00

X 1 2

-2.50

X 2 2

-6.78

X 3 2

改进的回归方程可用于计算变量范围内其他配方的预测响应值。

通过研究三维响应曲面图以评估配方变量对响应值的影响。图1为固定变量在预测最佳配方下,关键影响因素两两间相互作用对RIS-DMN成形性综合评分影响的可视化表示。

在二次方程描述自变量和响应的多项式方程中,当RIS-DMN成型性综合评分最高时,预测配方为:PVP K30 3.000 g,CS 2.569 g,溶剂含量54.806%。为便于实际操作,优选配方可表示为PVP K30-CS=1:0.86, 溶剂含量55%。

3.2 微针表征

3.2.1 表观形貌表征

以“3.1”项优选处方制备RIS-DMN,所得微针整体排列整齐,均匀分布在1.6 cm×1.6 cm的背衬上。微针扫描电子显微镜下的表观形态见图2,可见微针形态均一、表面光滑。通过电镜测量,单枚针体长度约为687.09 μm,底部直径约为481.97 μm,中心间距约为867.12 μm,是达到真皮层且不触碰毛细血管与神经末梢的理想形貌^[25]。

3.2.2 微针皮内溶解实验

结果见图3,随着针体在大鼠皮内停留时间的增加,其溶解程度逐渐增加。由图3可见,作用5 min时针体已出现明显的溶解;作用30 min后,微针针体几乎完全溶解,表明优选配方的RIS-DMN可较快溶解并滞留在大鼠皮肤内。

3.2.3 机械性能考察

为将药物通过皮肤途径有效输送进入全身循环,要求微针具备足够高的机械强度以抵达皮肤真皮层。微针穿刺情况见图4,在铝箔穿刺实验中,RIS-DMN可轻易刺穿铝箔并留下清晰可见的小孔;在离体大鼠皮肤穿刺实验中,经台盼蓝染色可见大鼠皮肤上清晰展现出与微针排列一致的蓝色小孔。结果说明,优选配方的RIS-DMN具有穿透铝箔和皮肤屏障的机械强度,符合实验要求。

3.2.4 皮肤屏障恢复实验

结果见图5。取下微针后可见大鼠腹部皮肤留有排列整齐的明显蓝色针孔,为微针作用后形成的孔道,表明台盼蓝已穿越皮肤屏障进入真皮层。取下微针 5 min后皮肤表面的蓝色针孔开始变淡,表明鼠皮已开始愈合;30 min后蓝色针孔大部分消失,同时台盼蓝出现明显晕色,表明由微针造成的细小孔洞已基本愈合,同时台盼蓝在真皮层中快速扩散;480 min后表皮蓝色针孔完全消失,台盼蓝颜色大幅淡化。皮肤孔道的闭合时间取决于皮肤屏障被破坏的程度,故根据蓝色针孔的变化可判断,配方优选的RIS-DMN给药后对皮肤的影响是可逆的,皮肤屏障功能可在短时间内得到恢复。

3.3 微针释放特性评价

3.3.1 针头载药量测定

本实验HPLC法峰分离性好、专属性强,具备良好的重现性与稳定性,在0.625~80.00 μg·mL^-1内展现出良好的线性关系(r²=0.999 7),可用于RIS的定量分析。阴性干扰实验结果见图6。经测量(n=3),高剂量RIS-DMN针头载药量为(420.83±18.09)μg,低剂量RIS-DMN针头载药量为(204.83±7.98)μg。

3.3.2 体外透皮实验

将优选处方的高、低剂量RIS-DMN分别刺入健康鼠皮并置于改良的Franz扩散池上进行体外透皮实验,以每个时间点的累积透过量作图,获得渗透曲线(图7)。由渗透曲线观察可知,高剂量RIS-DMN在3 h内迅速稳定渗透,随后逐渐减缓;低剂量RIS-DMN在6 h内稳定渗透,随后逐渐减缓。将累积透过量-时间数据经零级、一级和Higuchi模型拟合^[26],结果见表6,可见在2种含药浓度的微针组别中,一级动力学方程拟合模型的相关系数(r²)均为最高,因此可用该模型描述制得高、低剂量RIS-DMN在大鼠皮肤和生理盐水中的体外透过行为。

在体外透皮时间为3 h时,高剂量RIS-DMN的累积透过量为(459.11±17.09)μg,已高于“3.3.1”项下所测得针头载药量(420.83±18.09)μg,结合“3.2.2”项下微针快速溶解的特性,可认为0~3 h内微针针头迅速溶解并储留于大鼠真皮层,同时真皮层内的RIS以最高效率释放于接收液中,此时体外累积透过曲线斜率最大;随着RIS的不断释放,真皮层内含药量逐渐减少,累积透过曲线斜率下降。由于本实验所制备微针针体与背衬层均载有药物,在针头药物充分释放后,背衬层仍有RIS缓慢渗透,故24 h仍可见累积渗透量缓升。

3.4 药效学评价

3.4.1 血清生化指标测定

表7显示了RIS-DMN对卵巢摘除后骨质疏松症大鼠血清中Ca²⁺、P³⁺和ALP水平的影响。与假手术组相比,对照组大鼠血清Ca²⁺含量显著降低(P<0.01),此时给予高剂量RIS-DMN能够获得与灌胃给药相似的Ca²⁺水平回调效果(P<0.05),表明RIS给药可对抗卵巢摘除后骨质疏松症模型所引起的钙代谢紊乱。对照组与假手术组大鼠血清P³⁺水平有统计学差异,高剂量RIS-DMN呈现出一定的P³⁺调节能力(P<0.05)。有关研究表明^[27-28],ALP水平与机体骨密度呈负相关关系,鉴于此,可通过大鼠血清ALP变化以判定其骨质代谢状况。本研究中对照组ALP水平相比假手术组显著升高(P<0.01),表明卵巢摘除术可能诱导大鼠骨密度大幅下降。高、低剂量DMN与灌胃组大鼠ALP水平均有所回调,表明RIS给药能改善卵巢摘除后骨质疏松症大鼠的骨代谢,其中以高剂量RIS-DMN改善效果最为显著(P<0.01),表明经皮途径的RIS给药能够发挥出色的预防骨质疏松症作用。

3.4.2 骨密度测定

不同组别骨密度测量结果见图8。与假手术组相比,对照组大鼠骨密度显著降低(P<0.01),与“3.4.1”项下ALP显示结果一致。3个给药组别大鼠骨密度均有所提高(P<0.05),其中以高剂量微针组提升最为显著(P<0.01)。说明RIS-DMN对卵巢摘除骨质疏松症模型大鼠骨密度有优于灌胃给药的保护作用。

3.4.3 组织病理学

图9显示出RIS对大鼠骨微结构的实际影响。假手术组大鼠骨小梁数量丰富且较为粗大,局部相互连接,排列较规则,无骨细胞空腔,为健康的骨微结构。对照组大鼠骨小梁稀疏细小,排列不规则,多见脂肪细胞,为典型的骨质疏松病理结构,表明本实验卵巢摘除骨质疏松症模型构建成功,实验数据有参考意义。空白微针组大鼠骨微结构与对照组情况相似。低剂量微针组大鼠骨髓腔内骨小梁数量增加,骨小梁连接情况改善。高剂量微针组和灌胃组大鼠骨微结构均展现出较厚的骨组织皮质和丰富的髓腔骨小梁,相比之下,高剂量微针组中的骨小梁更为致密、形状更为饱满、尺寸和厚度更均匀、相互连接和排列更为规则,其髓腔充满骨髓细胞,已达到甚至优于正常大鼠的骨微结构;而灌胃组大鼠骨小梁排列不规则,髓腔内骨髓细胞较少。表明在卵巢摘除后骨质疏松症大鼠的骨组织保护中,RIS微针给药效果并不逊色于传统的灌胃给药,与“3.4.2”项下结果一致,展现出RIS经皮给药替代口服给药的巨大潜力。

4 讨论

收起

目前,口服双膦酸盐仍是治疗骨质疏松症的主要方法。然而苛刻的服用要求及严重的胃肠道副作用导致了其较差的临床依从性,这显然是不符合患者对当代药物便捷性、安全性需求的^{[11-13,29 -30]}。近年来,越来越多的研究试图解决口服双膦酸盐依从性差的问题,如使用纳米颗粒作为双膦酸盐的递送载体,可有效提高双膦酸盐的口服生物利用度^[31-32];开发双膦酸盐缓释制剂以解决服药前需禁食的限制^[33-34];减少口服双膦酸盐的剂量,以避免药物引起的胃肠道副作用等^[35]。尽管这些方法带来的改善是明确且直观的,但口服递药方式的保留意味着其生物利用度低、胃肠道副作用严重、服药体验差等问题无法从根源上被解决。若能够通过皮肤途径进行双膦酸盐给药,因口服引起胃肠道不良反应的问题即迎刃而解,患者不再需要通过禁食与维持直立姿势以减轻服药带来的胃肠道损伤,并能够轻松实现自行给药,这对于长期以传统口服双膦酸盐方式进行治疗的骨质疏松症患者而言,无疑是一种极大的解放^[36]。因此,本研究的重点在于双膦酸盐递送方式的转变。选用双膦酸盐代表性药物RIS为研究对象,探索并评价其通过皮肤途径进行递送的可行性。

皮肤屏障特殊的“砖墙结构”是高亲水性与大分子药物经皮进入体循环的主要障碍^[37]。近年来,微针技术逐渐成为该类药物经皮递送的重要突破口^[38]。借助微针针头,药物能够突破皮肤屏障直接释放于真皮层,并通过毛细血管吸收,从而发挥全身作用^[39]。为保证微针给药的依从性与有效性,通过提高DMN的皮内溶解速率,可缩短微针在体时间,使给药过程更为便捷舒适^[40];通过控制针体长度,可避免触及真皮层毛细血管和神经末梢,消除给药过程产生的疼痛^[41];足够的机械强度可保障药物的顺利递送;皮肤屏障的快速恢复可实现微针的长期用药。因此,表观形貌与机械性能、皮内溶解速率与皮肤屏障恢复情况被选为RIS-DMN表征的关键指标。

体外累积渗透结果显示(图7),本研究制备的高剂量RIS-DMN在3 h内透过大鼠皮肤进入接收液的药量超过微针针头含药量,表明配方优选后的RIS-DMN能够实现快速且高效的药物经皮递送,保证了RIS经皮给药的生物利用度^[42]。此外,本研究观察到高、低2种含药量的微针在体外透皮实验中均展现出与一级动力学较好的拟合效果(表6)。一级动力学体现药物被动转运的特点,在本实验中表明RIS的透皮行为更取决于药物的释放而非透过,进一步印证微针装载下的RIS能够顺利突破皮肤屏障并富集于真皮层中,发生顺浓度梯度的自由扩散行为。微针技术在亲水性大分子药物的经皮递送中展现出巨大潜力^[20]。

基金

收起

广州市科技计划项目(202206010188)
广州中医药大学“双一流”与高水平大学学科协同创新团队培育项目(2021xk78)

参考文献

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2024年第59卷第16期

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文章信息

doi: 10.11669/cpj.2024.16.010

接收时间：2023-02-19
首发时间：2025-10-29
出版时间：2024-08-22

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文章信息

作者

出版历史

收稿日期：2023-02-19

基金

广州市科技计划项目(202206010188)

广州中医药大学“双一流”与高水平大学学科协同创新团队培育项目(2021xk78)

作者信息

¹ 广州中医药大学中药学院, 广州 510006

² 帝国理工学院生命科学与医学学院,伦敦 SW72AZ

通讯作者:

* 王利胜,男,博士,教授,博士生导师研究方向:药物制剂新技术与新剂型 Tel:(020)39358290

参考文献

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https://castjournals.cast.org.cn/joweb/zgyxzz/CN/10.11669/cpj.2024.16.010

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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TxT

Independent variables	Coded levels
X₁:PVP K30/g	1	2	3
X₂:CS/g	1	2	3
X₃:Liquid/%	45	55	65

Independent
variables

Coded levels

-1

X₁:PVP K30/g

X₂:CS/g

X₃:Liquid/%

Project	Standard for evaluation
Microneedle body morphology	Severe bending or majority fracture (0-3 points). Bending or a few breaks (4-10 points). Slightly curved, does not affect the use (11-13 points). No bending and no breaking (14-15 min)
Film-formation	Can not form film, after drying in the mold is broken slag (0-2 points). After drying and curing, it becomes film-like, has severe shrinkage or is easy to break (3-5 minutes). Slightly crumpled or cracked after drying and curing, but does not affect the use (6-10 points). After drying and curing, a complete array of microneedles of moderate thickness is formed (11-15 min)
Tip hardness	Can not Pierce aluminum foil (0-1 score). It could puncture aluminum foil but could not puncture rat skin, and the tip of the needle broke (2-5 points). Can puncture aluminum foil and rat skin without breaking the tip (6-10 minutes)
Backing performance	The backing is obviously crumpled, and the pressure is fragile (0-2 points). The backing has no obvious shrinkage, but presents a curved bulge, and is fragile (3-6 points) under excessive pressure. Flat backing without wrinkling, pressure is not easy to break (7-10 points)
Number of bubbles	Filled with a large number of bubbles or forming many empty needles, so that microneedles cannot be used (0-1 score). There are a few bubbles or slightly empty needles (2-5 points). There is a small amount of bubbles but it does not affect the use (6-9 points). No visible bubbles (10 points)
Microneedle body integrity rate	A large number of needle bodies are missing, making microneedles unusable (0-1 score). Half of the needle body is missing (2-3 points). A small amount of needle body missing, does not affect the use of (4-9 points). Absence of needle body (10 points)

Project

Standard for evaluation

Microneedle body
morphology

Severe bending or majority fracture (0-3 points). Bending or a few breaks (4-10 points). Slightly curved, does not affect the use (11-13 points). No bending and no breaking (14-15 min)

Film-formation

Can not form film, after drying in the mold is broken slag (0-2 points). After drying and curing, it becomes film-like, has severe shrinkage or is easy to break (3-5 minutes). Slightly crumpled or cracked after drying and curing, but does not affect the use (6-10 points). After drying and curing, a complete array of microneedles of moderate thickness is formed (11-15 min)

Tip hardness

Can not Pierce aluminum foil (0-1 score). It could puncture aluminum foil but could not puncture rat skin, and the tip of the needle broke (2-5 points). Can puncture aluminum foil and rat skin without breaking the tip (6-10 minutes)

Backing performance

The backing is obviously crumpled, and the pressure is fragile (0-2 points). The backing has no obvious shrinkage, but presents a curved bulge, and is fragile (3-6 points) under excessive pressure. Flat backing without wrinkling, pressure is not easy to break (7-10 points)

Number of bubbles

Filled with a large number of bubbles or forming many empty needles, so that microneedles cannot be used (0-1 score). There are a few bubbles or slightly empty needles (2-5 points). There is a small amount of bubbles but it does not affect the use (6-9 points). No visible bubbles (10 points)

Microneedle body
integrity rate

A large number of needle bodies are missing, making microneedles unusable (0-1 score). Half of the needle body is missing (2-3 points). A small amount of needle body missing, does not affect the use of (4-9 points). Absence of needle body (10 points)

Run	Variables	Responses
1	1	2	65	13	8	9	8	6	10	54
2	3	2	55	13	12	7	7	6	6	51
3	3	3	65	14	10	8	9	5	6	52
4	3	1	55	13	12	7	8	5	4	49
5	2	3	55	10	7	8	4	7	6	42
6	1	3	45	13	11	6	8	7	9	54
7	3	1	65	12	12	8	8	6	9	55
8	1	1	45	14	10	8	9	9	10	60
9	1	3	65	13	8	10	8	8	8	55
10	2	1	45	13	12	8	7	10	7	57
11	2	2	55	10	14	4	8	10	10	56
12	2	3	65	12	7	6	5	9	9	48
13	1	2	55	12	14	5	8	9	10	58
14	3	2	65	14	13	8	8	10	10	63
15	2	1	65	13	8	8	6	10	9	54
16	1	3	55	12	6	4	6	8	9	45
17	3	1	45	8	11	6	7	6	10	48
18	2	3	45	12	12	1	6	9	10	50
19	3	2	45	10	7	3	5	5	9	39
20	1	2	45	10	12	4	8	9	9	52
21	2	1	55	10	12	7	9	8	9	55

Run

Variables

Responses

PVP K30
X₁/g

CS
X₂/g

Liquid
X₃/%

Microneedle body
morphology(15)

film-formation
(15)

Tip hardness
(10)

Backing
performance(10)

Number of
bubbles(10)

Microneedle body
integrity rate(10)

Total points
(70)

Model	SD	CV/%	r²	Adjusted R²	Predicted R²	PRESS	Result
Linear	5.14	9.83	0.324 5	0.205 3	-0.010 5	670.80
2FI	4.46	8.54	0.580 6	0.400 9	0.042 2	635.76
Quadratic	0.969 0	1.85	0.984 4	0.971 7	0.932 6	44.76	Suggested
Cubic	0.802 8	1.54	0.995 1	0.980 6	0.858 8	93.76	Aliased

Model

CV/%

r²

Adjusted R²

Predicted R²

PRESS

Result

Linear

5.14

9.83

0.324 5

0.205 3

-0.010 5

670.80

2FI

4.46

8.54

0.580 6

0.400 9

0.042 2

635.76

Quadratic

0.969 0

1.85

0.984 4

0.971 7

0.932 6

44.76

Suggested

Cubic

0.802 8

1.54

0.995 1

0.980 6

0.858 8

93.76

Aliased

Source	df	Coefficient estimate	Sum of squares	Mean square	F	P
Model	9		653.48	72.61	77.33	<0.000 1
Intercept	1	60.44
A-PVP K30	1	3.26	131.32	131.32	139.86	<0.000 1
B-CS	1	-1.24	19.1	19.1	20.34	0.000 9
C-Liquid	1	-1.29	21.89	21.89	23.32	0.000 5
AB	1	4.06	59.21	59.21	63.06	<0.000 1
AC	1	1.11	7.84	7.84	8.35	0.014 7
BC	1	-0.222 2	0.313 7	0.313 7	0.334 1	0.574 9
A²	1	-2.00	11.37	11.37	12.11	0.005 2
B²	1	-2.50	17.76	17.76	18.92	0.001 2
C²	1	-6.78	190.82	190.82	203.23	<0.000 1
Residual	11		10.33	0.938 9
Cor Total	20		663.81

Source

Coefficient estimate

Sum of squares

Mean square

Model

653.48

72.61

77.33

<0.000 1

Intercept

60.44

A-PVP K30

3.26

131.32

139.86

<0.000 1

B-CS

-1.24

19.1

20.34

0.000 9

C-Liquid

-1.29

21.89

23.32

0.000 5

4.06

59.21

63.06

<0.000 1

1.11

7.84

8.35

0.014 7

-0.222 2

0.313 7

0.334 1

0.574 9

A²

-2.00

11.37

12.11

0.005 2

B²

-2.50

17.76

18.92

0.001 2

C²

-6.78

190.82

203.23

<0.000 1

Residual

10.33

0.938 9

Cor Total

663.81

Group	Model	Equation	Parameter	r²
High-dose	zero-order	F=k₀·t	k₀=9.670	0.482 3
	first-order	F=M·[1-Exp(-k₁·t)]	k₁=0.418, M=322.22	0.965 9
	Higuchi	F=k_H·t^0.5	k_H=64.555	0.680 8
Low-dose	zero-order	F=k₀·t	k₀=6.751	0.819 3
	first-order	F=M·[1-Exp(-k₁·t)]	k₁=0.139, M=169.36	0.991 2
	Higuchi	F=k_H·t^0.5	k_H=40.812	0.948 2

Group

Model

Equation

Parameter

r²

High-dose

zero-order

F=k₀·t

k₀=9.670

0.482 3

first-order

F=M·[1-Exp(-k₁·t)]

k₁=0.418, M=322.22

0.965 9

Higuchi

F=k_H·t^0.5

k_H=64.555

0.680 8

Low-dose

zero-order

F=k₀·t

k₀=6.751

0.819 3

first-order

F=M·[1-Exp(-k₁·t)]

k₁=0.139, M=169.36

0.991 2

Higuchi

F=k_H·t^0.5

k_H=40.812

0.948 2

Group	Ca²⁺	P³⁺	ALP
Sham	2.291±0.042²⁾	1.565±0.154¹⁾	20.596±8.549²⁾
Control	2.063±0.037⁴⁾	1.411±0.159³⁾	42.393±13.387⁴⁾
DMN-Blank	2.045±0.037⁴⁾	1.555±0.075	38.922±6.992⁴⁾
DMN-Low	2.067±0.056⁴⁾	1.535±0.063	30.610±4.701¹⁾
DMN-High	2.140±0.037¹⁾⁴⁾	1.408±0.082³⁾	23.526±4.904²⁾
Gavage	2.173±0.069²⁾⁴⁾	1.496±0.107	27.138±6.039²⁾

Group

Ca²⁺

P³⁺

ALP

Sham

2.291±0.042²⁾

1.565±0.154¹⁾

20.596±8.549²⁾

Control

2.063±0.037⁴⁾

1.411±0.159³⁾

42.393±13.387⁴⁾

DMN-Blank

2.045±0.037⁴⁾

1.555±0.075

38.922±6.992⁴⁾

DMN-Low

2.067±0.056⁴⁾

1.535±0.063

30.610±4.701¹⁾

DMN-High

2.140±0.037¹⁾⁴⁾

1.408±0.082³⁾

23.526±4.904²⁾

Gavage

2.173±0.069²⁾⁴⁾

1.496±0.107

27.138±6.039²⁾