药学学报

Element	Quality attribute	Target	Justification
QTPP	Route of administration	Intravenous	Increase the bioavailability of active substances at the tumor site
Dosage form	Liposome	Co-encapsulate and co-delivery of Om and As
Outer packing	Penicillin bottle	Ensure stability of Om-As-Lip during storage
Prolonged blood circulation time	PEGylated liposomes	To avoid reticuloendothelial system macrophages
Appearance	Clear and transparent	Quality standards for injections
In vitro release	Slow release	Standards for sustained-release preparations
CQAs	EE%	50%-100%	Reduced drug loss and production costs
Size	100-200 nm	Produce EPR effect and enhance drug concentration
PDI	< 0.3	To predict the behavior of Om-As-Lip in vivo
Zeta potential	≤ -20 mV / ≥ 20 mV	To enhance the stability
As∶Om	1∶1-1∶2	To achieve the desired effect at the tumor site

Element

Quality attribute

Target

Justification

QTPP

Route of administration

Intravenous

Increase the bioavailability of active substances at the tumor site

Dosage form

Liposome

Co-encapsulate and co-delivery of Om and As

Outer packing

Penicillin bottle

Ensure stability of Om-As-Lip during storage

Prolonged blood circulation time

PEGylated liposomes

To avoid reticuloendothelial system macrophages

Appearance

Clear and transparent

Quality standards for injections

In vitro release

Slow release

Standards for sustained-release preparations

CQAs

EE%

50%-100%

Reduced drug loss and production costs

Size

100-200 nm

Produce EPR effect and enhance drug concentration

PDI

< 0.3

To predict the behavior of Om-As-Lip in vivo

Zeta potential

≤ -20 mV / ≥ 20 mV

To enhance the stability

As∶Om

1∶1-1∶2

To achieve the desired effect at the tumor site

Element	Quality attribute	Target	Justification
QTPP	Route of administration	Intravenous	Increase the bioavailability of active substances at the tumor site
Dosage form	Liposome	Co-encapsulate and co-delivery of Om and As
Outer packing	Penicillin bottle	Ensure stability of Om-As-Lip during storage
Prolonged blood circulation time	PEGylated liposomes	To avoid reticuloendothelial system macrophages
Appearance	Clear and transparent	Quality standards for injections
In vitro release	Slow release	Standards for sustained-release preparations
CQAs	EE%	50%-100%	Reduced drug loss and production costs
Size	100-200 nm	Produce EPR effect and enhance drug concentration
PDI	< 0.3	To predict the behavior of Om-As-Lip in vivo
Zeta potential	≤ -20 mV / ≥ 20 mV	To enhance the stability
As∶Om	1∶1-1∶2	To achieve the desired effect at the tumor site

Element

Quality attribute

Target

Justification

QTPP

Route of administration

Intravenous

Increase the bioavailability of active substances at the tumor site

Dosage form

Liposome

Co-encapsulate and co-delivery of Om and As

Outer packing

Penicillin bottle

Ensure stability of Om-As-Lip during storage

Prolonged blood circulation time

PEGylated liposomes

To avoid reticuloendothelial system macrophages

Appearance

Clear and transparent

Quality standards for injections

In vitro release

Slow release

Standards for sustained-release preparations

CQAs

EE%

50%-100%

Reduced drug loss and production costs

Size

100-200 nm

Produce EPR effect and enhance drug concentration

PDI

< 0.3

To predict the behavior of Om-As-Lip in vivo

Zeta potential

≤ -20 mV / ≥ 20 mV

To enhance the stability

As∶Om

1∶1-1∶2

To achieve the desired effect at the tumor site

Parameter	Failure mode	Failure effect	Potential cause	Control method	S	D	O	RPN
EE/%	Drug leakage	Increase administration frequency	Phospholipids concentration	Confirm the appropriate concentration range	4	3	4	48
As concentration	4	3	4	48
Om concentration	4	3	3	48
Cholesterol concentration	4	3	3	36
Synthetic phospholipids ratio	Optimizing the phospholipid ratio	4	3	4	48
Om loading method	pH gradient	3	1	2	6
The pH of the hydration medium	Optimizing the citric acid concentration	4	2	4	32
Incubation time	Optimizing incubation time	4	2	3	24
Size, PDI	Large size distribution	Reduced stability	Uniform particle size	Magnetic stirring speed	2	1	3	6
Complex process in vivo	Ultrasound time	2	1	3	6
Zeta potential	> -20 mV	Aggregation during storage life	Types of phospholipids	Use highly charged phospholipid	2	1	5	10
Phospholipids concentration	Optimize phospholipids concentration	4	3	4	48
Prolong blood circulation time	Eliminated by the RES system	Reduced effectiveness of treatment	PEG modified liposomes	Optimize DSPE-mPEG₂₀₀₀ concentration	3	2	3	18
Increased side effects
Safety	Residual organic solvent	Increased side effects	Volume of absolute ethanol	Optimize the volume of absolute ethanol	4	3	3	36
Reduced storage stability

Parameter

Failure mode

Failure effect

Potential cause

Control method

RPN

EE/%

Drug leakage

Increase administration frequency

Phospholipids concentration

Confirm the appropriate concentration range

As concentration

Om concentration

Cholesterol concentration

Synthetic phospholipids ratio

Optimizing the phospholipid ratio

Om loading method

pH gradient

The pH of the hydration medium

Optimizing the citric acid concentration

Incubation time

Optimizing incubation time

Size, PDI

Large size distribution

Reduced stability

Uniform particle size

Magnetic stirring speed

Complex process in vivo

Ultrasound time

Zeta potential

> -20 mV

Aggregation during storage life

Types of phospholipids

Use highly charged phospholipid

Phospholipids concentration

Optimize phospholipids concentration

Prolong blood circulation time

Eliminated by the RES system

Reduced effectiveness of treatment

PEG modified liposomes

Optimize DSPE-mPEG₂₀₀₀ concentration

Increased side effects

Safety

Residual organic solvent

Increased side effects

Volume of absolute ethanol

Optimize the volume of absolute ethanol

Reduced storage stability

Parameter	Failure mode	Failure effect	Potential cause	Control method	S	D	O	RPN
EE/%	Drug leakage	Increase administration frequency	Phospholipids concentration	Confirm the appropriate concentration range	4	3	4	48
As concentration	4	3	4	48
Om concentration	4	3	3	48
Cholesterol concentration	4	3	3	36
Synthetic phospholipids ratio	Optimizing the phospholipid ratio	4	3	4	48
Om loading method	pH gradient	3	1	2	6
The pH of the hydration medium	Optimizing the citric acid concentration	4	2	4	32
Incubation time	Optimizing incubation time	4	2	3	24
Size, PDI	Large size distribution	Reduced stability	Uniform particle size	Magnetic stirring speed	2	1	3	6
Complex process in vivo	Ultrasound time	2	1	3	6
Zeta potential	> -20 mV	Aggregation during storage life	Types of phospholipids	Use highly charged phospholipid	2	1	5	10
Phospholipids concentration	Optimize phospholipids concentration	4	3	4	48
Prolong blood circulation time	Eliminated by the RES system	Reduced effectiveness of treatment	PEG modified liposomes	Optimize DSPE-mPEG₂₀₀₀ concentration	3	2	3	18
Increased side effects
Safety	Residual organic solvent	Increased side effects	Volume of absolute ethanol	Optimize the volume of absolute ethanol	4	3	3	36
Reduced storage stability

Parameter

Failure mode

Failure effect

Potential cause

Control method

RPN

EE/%

Drug leakage

Increase administration frequency

Phospholipids concentration

Confirm the appropriate concentration range

As concentration

Om concentration

Cholesterol concentration

Synthetic phospholipids ratio

Optimizing the phospholipid ratio

Om loading method

pH gradient

The pH of the hydration medium

Optimizing the citric acid concentration

Incubation time

Optimizing incubation time

Size, PDI

Large size distribution

Reduced stability

Uniform particle size

Magnetic stirring speed

Complex process in vivo

Ultrasound time

Zeta potential

> -20 mV

Aggregation during storage life

Types of phospholipids

Use highly charged phospholipid

Phospholipids concentration

Optimize phospholipids concentration

Prolong blood circulation time

Eliminated by the RES system

Reduced effectiveness of treatment

PEG modified liposomes

Optimize DSPE-mPEG₂₀₀₀ concentration

Increased side effects

Safety

Residual organic solvent

Increased side effects

Volume of absolute ethanol

Optimize the volume of absolute ethanol

Reduced storage stability

Run	X₁	X₂	X₃/mg·mL^-1	X₄	X₅	X₆	X₇/mL	X₈	X₉/h	Y₁/%
PB-1	1 (-1)	3 (1)	0.6 (1)	12 (1)	1 (-1)	11∶1 (-1)	2 (-1)	5 (1)	0.5 (-1)	156.94
PB-2	1 (-1)	1 (-1)	0.6 (1)	3 (-1)	4 (1)	7∶1 (1)	2 (-1)	5 (1)	3 (1)	140.90
PB-3	3 (1)	1 (-1)	0.6 (1)	12 (1)	4 (1)	11∶1 (-1)	2 (-1)	2 (-1)	3 (1)	101.94
PB-4	3 (1)	1 (-1)	0.3 (-1)	3 (-1)	4 (1)	11∶1 (-1)	5 (1)	5 (1)	0.5 (-1)	142.80
PB-5	1 (-1)	1 (-1)	0.3 (-1)	3 (-1)	1 (-1)	11∶1 (-1)	2 (-1)	2 (-1)	0.5 (-1)	151.59
PB-6	1 (-1)	1 (-1)	0.3 (-1)	12 (1)	1 (-1)	7∶1 (1)	5 (1)	2 (-1)	3 (1)	95.11
PB-7	1 (-1)	3 (1)	0.3 (-1)	12 (1)	4 (1)	11∶1 (-1)	5 (1)	5 (1)	3 (1)	139.22
PB-8	3 (1)	1 (-1)	0.6 (1)	12 (1)	1 (-1)	7∶1 (1)	5 (1)	5 (1)	0.5 (-1)	100.94
PB-9	3 (1)	3 (1)	0.3 (-1)	12 (1)	4 (1)	7∶1 (1)	2 (-1)	2 (-1)	0.5 (-1)	66.13
PB-10	3 (1)	3 (1)	0.6 (1)	3 (-1)	1 (-1)	11∶1 (-1)	5 (1)	2 (-1)	3 (1)	118.52
PB-11	1 (-1)	3 (1)	0.6 (1)	3 (-1)	4 (1)	7∶1 (1)	5 (1)	2 (-1)	0.5 (-1)	151.52
PB-12	3 (1)	3 (1)	0.3 (-1)	3 (-1)	1 (-1)	7∶1 (1)	2 (-1)	5 (1)	3 (1)	93.92

Run

X₁

X₂

X₃/mg·mL^-1

X₄

X₅

X₆

X₇/mL

X₈

X₉/h

Y₁/%

PB-1

1 (-1)

3 (1)

0.6 (1)

12 (1)

1 (-1)

11∶1 (-1)

2 (-1)

5 (1)

0.5 (-1)

156.94

PB-2

1 (-1)

0.6 (1)

3 (-1)

4 (1)

7∶1 (1)

2 (-1)

5 (1)

3 (1)

140.90

PB-3

3 (1)

1 (-1)

0.6 (1)

12 (1)

4 (1)

11∶1 (-1)

2 (-1)

3 (1)

101.94

PB-4

3 (1)

1 (-1)

0.3 (-1)

3 (-1)

4 (1)

11∶1 (-1)

5 (1)

0.5 (-1)

142.80

PB-5

1 (-1)

0.3 (-1)

3 (-1)

1 (-1)

11∶1 (-1)

2 (-1)

0.5 (-1)

151.59

PB-6

1 (-1)

0.3 (-1)

12 (1)

1 (-1)

7∶1 (1)

5 (1)

2 (-1)

3 (1)

95.11

PB-7

1 (-1)

3 (1)

0.3 (-1)

12 (1)

4 (1)

11∶1 (-1)

5 (1)

3 (1)

139.22

PB-8

3 (1)

1 (-1)

0.6 (1)

12 (1)

1 (-1)

7∶1 (1)

5 (1)

0.5 (-1)

100.94

PB-9

3 (1)

0.3 (-1)

12 (1)

4 (1)

7∶1 (1)

2 (-1)

0.5 (-1)

66.13

PB-10

3 (1)

0.6 (1)

3 (-1)

1 (-1)

11∶1 (-1)

5 (1)

2 (-1)

3 (1)

118.52

PB-11

1 (-1)

3 (1)

0.6 (1)

3 (-1)

4 (1)

7∶1 (1)

5 (1)

2 (-1)

0.5 (-1)

151.52

PB-12

3 (1)

0.3 (-1)

3 (-1)

1 (-1)

7∶1 (1)

2 (-1)

5 (1)

3 (1)

93.92

Run	X₁	X₂	X₃/mg·mL^-1	X₄	X₅	X₆	X₇/mL	X₈	X₉/h	Y₁/%
PB-1	1 (-1)	3 (1)	0.6 (1)	12 (1)	1 (-1)	11∶1 (-1)	2 (-1)	5 (1)	0.5 (-1)	156.94
PB-2	1 (-1)	1 (-1)	0.6 (1)	3 (-1)	4 (1)	7∶1 (1)	2 (-1)	5 (1)	3 (1)	140.90
PB-3	3 (1)	1 (-1)	0.6 (1)	12 (1)	4 (1)	11∶1 (-1)	2 (-1)	2 (-1)	3 (1)	101.94
PB-4	3 (1)	1 (-1)	0.3 (-1)	3 (-1)	4 (1)	11∶1 (-1)	5 (1)	5 (1)	0.5 (-1)	142.80
PB-5	1 (-1)	1 (-1)	0.3 (-1)	3 (-1)	1 (-1)	11∶1 (-1)	2 (-1)	2 (-1)	0.5 (-1)	151.59
PB-6	1 (-1)	1 (-1)	0.3 (-1)	12 (1)	1 (-1)	7∶1 (1)	5 (1)	2 (-1)	3 (1)	95.11
PB-7	1 (-1)	3 (1)	0.3 (-1)	12 (1)	4 (1)	11∶1 (-1)	5 (1)	5 (1)	3 (1)	139.22
PB-8	3 (1)	1 (-1)	0.6 (1)	12 (1)	1 (-1)	7∶1 (1)	5 (1)	5 (1)	0.5 (-1)	100.94
PB-9	3 (1)	3 (1)	0.3 (-1)	12 (1)	4 (1)	7∶1 (1)	2 (-1)	2 (-1)	0.5 (-1)	66.13
PB-10	3 (1)	3 (1)	0.6 (1)	3 (-1)	1 (-1)	11∶1 (-1)	5 (1)	2 (-1)	3 (1)	118.52
PB-11	1 (-1)	3 (1)	0.6 (1)	3 (-1)	4 (1)	7∶1 (1)	5 (1)	2 (-1)	0.5 (-1)	151.52
PB-12	3 (1)	3 (1)	0.3 (-1)	3 (-1)	1 (-1)	7∶1 (1)	2 (-1)	5 (1)	3 (1)	93.92

Run

X₁

X₂

X₃/mg·mL^-1

X₄

X₅

X₆

X₇/mL

X₈

X₉/h

Y₁/%

PB-1

1 (-1)

3 (1)

0.6 (1)

12 (1)

1 (-1)

11∶1 (-1)

2 (-1)

5 (1)

0.5 (-1)

156.94

PB-2

1 (-1)

0.6 (1)

3 (-1)

4 (1)

7∶1 (1)

2 (-1)

5 (1)

3 (1)

140.90

PB-3

3 (1)

1 (-1)

0.6 (1)

12 (1)

4 (1)

11∶1 (-1)

2 (-1)

3 (1)

101.94

PB-4

3 (1)

1 (-1)

0.3 (-1)

3 (-1)

4 (1)

11∶1 (-1)

5 (1)

0.5 (-1)

142.80

PB-5

1 (-1)

0.3 (-1)

3 (-1)

1 (-1)

11∶1 (-1)

2 (-1)

0.5 (-1)

151.59

PB-6

1 (-1)

0.3 (-1)

12 (1)

1 (-1)

7∶1 (1)

5 (1)

2 (-1)

3 (1)

95.11

PB-7

1 (-1)

3 (1)

0.3 (-1)

12 (1)

4 (1)

11∶1 (-1)

5 (1)

3 (1)

139.22

PB-8

3 (1)

1 (-1)

0.6 (1)

12 (1)

1 (-1)

7∶1 (1)

5 (1)

0.5 (-1)

100.94

PB-9

3 (1)

0.3 (-1)

12 (1)

4 (1)

7∶1 (1)

2 (-1)

0.5 (-1)

66.13

PB-10

3 (1)

0.6 (1)

3 (-1)

1 (-1)

11∶1 (-1)

5 (1)

2 (-1)

3 (1)

118.52

PB-11

1 (-1)

3 (1)

0.6 (1)

3 (-1)

4 (1)

7∶1 (1)

5 (1)

2 (-1)

0.5 (-1)

151.52

PB-12

3 (1)

0.3 (-1)

3 (-1)

1 (-1)

7∶1 (1)

2 (-1)

5 (1)

3 (1)

93.92

Run	Level		Factor		Result
1	-1	0	1		1	10	51∶9		93.13	49.72
2	0	0	0	2	10	52.5∶7.5	89.87	42.34
3	1	0	1	3	10	51∶9	62.88	31.31
4	0	-1	-1	2	8	54∶6	80.57	27.28
5	1	-1	0	3	8	52.5∶7.5	58.02	32.82
6	1	0	-1	3	10	54∶6	70.80	41.01
7	0	0	0	2	10	52.5∶7.5	98.49	35.91
8	-1	1	0	1	12	52.5∶7.5	86.43	63.79
9	0	1	1	2	12	51∶9	91.38	59.14
10	-1	-1	0	1	8	52.5∶7.5	96.69	26.40
11	0	-1	1	2	8	51∶9	93.99	35.63
12	0	0	0	2	10	52.5∶7.5	94.52	39.78
13	0	0	0	2	10	52.5∶7.5	93.53	31.85
14	1	1	0	3	12	52.5∶7.5	56.41	30.33
15	-1	0	1	1	10	51∶9	93.94	43.96
16	0	1	1	2	12	51∶9	100.0	47.47
17	0	0	0	2	10	52.5∶7.5	91.78	33.04

Run

Level

Factor

Result

X₁

X₂

X₃

X₁

X₂

X₃

Y₁/%

Y₂/%

-1

51∶9

93.13

49.72

52.5∶7.5

89.87

42.34

51∶9

62.88

31.31

-1

54∶6

80.57

27.28

-1

52.5∶7.5

58.02

32.82

-1

54∶6

70.80

41.01

52.5∶7.5

98.49

35.91

-1

52.5∶7.5

86.43

63.79

51∶9

91.38

59.14

-1

52.5∶7.5

96.69

26.40

-1

51∶9

93.99

35.63

52.5∶7.5

94.52

39.78

52.5∶7.5

93.53

31.85

52.5∶7.5

56.41

30.33

-1

51∶9

93.94

43.96

51∶9

100.0

47.47

52.5∶7.5

91.78

33.04

Run	Level		Factor		Result
1	-1	0	1		1	10	51∶9		93.13	49.72
2	0	0	0	2	10	52.5∶7.5	89.87	42.34
3	1	0	1	3	10	51∶9	62.88	31.31
4	0	-1	-1	2	8	54∶6	80.57	27.28
5	1	-1	0	3	8	52.5∶7.5	58.02	32.82
6	1	0	-1	3	10	54∶6	70.80	41.01
7	0	0	0	2	10	52.5∶7.5	98.49	35.91
8	-1	1	0	1	12	52.5∶7.5	86.43	63.79
9	0	1	1	2	12	51∶9	91.38	59.14
10	-1	-1	0	1	8	52.5∶7.5	96.69	26.40
11	0	-1	1	2	8	51∶9	93.99	35.63
12	0	0	0	2	10	52.5∶7.5	94.52	39.78
13	0	0	0	2	10	52.5∶7.5	93.53	31.85
14	1	1	0	3	12	52.5∶7.5	56.41	30.33
15	-1	0	1	1	10	51∶9	93.94	43.96
16	0	1	1	2	12	51∶9	100.0	47.47
17	0	0	0	2	10	52.5∶7.5	91.78	33.04

Run

Level

Factor

Result

X₁

X₂

X₃

X₁

X₂

X₃

Y₁/%

Y₂/%

-1

51∶9

93.13

49.72

52.5∶7.5

89.87

42.34

51∶9

62.88

31.31

-1

54∶6

80.57

27.28

-1

52.5∶7.5

58.02

32.82

-1

54∶6

70.80

41.01

52.5∶7.5

98.49

35.91

-1

52.5∶7.5

86.43

63.79

51∶9

91.38

59.14

-1

52.5∶7.5

96.69

26.40

-1

51∶9

93.99

35.63

52.5∶7.5

94.52

39.78

52.5∶7.5

93.53

31.85

52.5∶7.5

56.41

30.33

-1

51∶9

93.94

43.96

51∶9

100.0

47.47

52.5∶7.5

91.78

33.04

基于质量源于设计(QbD) 理念的氧化苦参碱-黄芪甲苷共载脂质体处方工艺研究

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韦良银 ¹^,² , 李霞 ¹^,² , 王虹 ¹^,² , 黄琳清 ¹^,² , 刘聪燕 ¹^,²^,^* , 陈彦 ¹^,²^,^*

药学学报 | 研究论文 2024,59(1): 232-242

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药学学报 | 研究论文 2024, 59(1): 232-242

基于质量源于设计(QbD) 理念的氧化苦参碱-黄芪甲苷共载脂质体处方工艺研究

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韦良银¹^,², 李霞¹^,², 王虹¹^,², 黄琳清¹^,², 刘聪燕¹^,²^,^*, 陈彦¹^,²^,^*

作者信息

1.南京中医药大学附属中西医结合医院, 江苏南京 210028

2.江苏省中医药研究院, 中药组分与微生态研究中心, 江苏南京 210028

通讯作者:

^*刘聪燕，Tel: 86-25-52362155, E-mail: liucongyan2007@126.com;

陈彦, E-mail: ychen2020@hotmail.com

Formulation and technology of oxymatrine-astragaloside Ⅳ coloaded liposomes based on quality by design

Liang-yin WEI¹^,², Xia LI¹^,², Hong WANG¹^,², Lin-qing HUANG¹^,², Cong-yan LIU¹^,²^,^*, Yan CHEN¹^,²^,^*

Affiliations

1. Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing 210028, China

2. Multi-component of Traditional Chinese Medicine and Microecology Research Center, Jiangsu Provincial Academy of Chinese Medicine, Nanjing 210028, China

出版时间: 2024-01-12 doi: 10.16438/j.0513-4870.2023-0515

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摘要

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本研究以“质量源于设计” (quality by design, QbD) 理念为核心, 优化氧化苦参碱-黄芪甲苷共载脂质体(oxymatrine-astragaloside Ⅳ liposomes, Om-As-Lip) 的处方工艺并对其进行放大验证。采用乙醇注入联合pH梯度法制备Om-As-Lip, 通过双重风险评估工具、Plackett-Burman设计和Box-Behnken响应面实验对其关键物料属性进行优化, 建立设计空间; 进一步考察Om-As-Lip的放大工艺, 采用单因素试验优化高压匀质的关键工艺参数, 并对其终产品进行质量评价。研究结果发现, 黄芪甲苷药脂比、胆脂比和混合磷脂比例(氢化大豆卵磷脂∶大豆卵磷脂) 是影响Om-As-Lip质量的关键材料属性, Box-Behnken设计建立的回归模型具有良好的预测性, 并确定Om-As-Lip的最佳处方为: 黄芪甲苷药脂比为1∶40, 胆脂比为1∶10, 氢化大豆卵磷脂∶大豆卵磷脂为51∶9。设计空间内的胆脂比可控制在1∶12~1∶5, 氢化大豆卵磷脂∶大豆卵磷脂比例可控制在1∶7~17∶3。Om-As-Lip高压匀质的最佳压力为600 bar, 循环次数为6次, 温度为4 ℃。基于QbD理念制备的Om-As-Lip质量评价符合预期, 建立的处方工艺稳定可行, 有望为其今后的开发应用奠定实验基础。

关键词

质量源于设计 / 脂质体 / 高压匀质 / 黄芪甲苷 / 氧化苦参碱

Abstract

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To optimize the formulation and technology of oxymatrine-astragaloside Ⅳ coloaded liposomes (Om-As-Lip) based on quality by design (QbD) principles, and further to verify the feasibility of its amplification process, Om-As-Lip was prepared by ethanol injection combined with pH gradient method. The critical material attributions of Om-As-Lip were evaluated by dual-risk analysis tools and Plackett-Burman design (PBD). The formulation of Om-As-Lip was further optimized with the Box-Behnken design (BBD). The design space was also established based on the contour plots of BBD. In order to further investigate the amplification process of Om-As-Lip, the critical process parameters of high-pressure homogenization (HPH) were optimized by single-factor test, and the quality of the final product was also evaluated. The results of risk analysis and PBD confirmed that the astragaloside concentration, cholesterol concentration, and phospholipid ratio (HSPC∶SPC) were the ctitical material attributes. The model established by BBD had a good predictability, and the optimized mass ratio of As to phospholipids was 1∶40, cholesterol to phospholipids was 1∶10, HSPC to SPC was 51∶9. The design space of Om-As-Lip was as follows: the ratio of cholesterol to phospholipids was 1∶12-1∶5 and HSPC to SPC was 1∶7-17∶3. The optimized high-pressure homogenization pressure was 600 bar, temperature was 4 ℃, and cycle times was 6 times for HPH-Om-As-Lip. The quality of Om-As-Lip prepared based on the QbD concept can meet the expected CQAs, and the formulation and technology established can provide a reliable experimental basis for its future development and applications.

Key words

quality by design / liposome / high-pressure homogenization / astragaloside Ⅳ / oxymatrine

引用本文

韦良银, 李霞, 王虹, 黄琳清, 刘聪燕, 陈彦. 基于质量源于设计(QbD) 理念的氧化苦参碱-黄芪甲苷共载脂质体处方工艺研究. 药学学报, 2024 , 59 (1) : 232 -242 . DOI: 10.16438/j.0513-4870.2023-0515

Liang-yin WEI, Xia LI, Hong WANG, Lin-qing HUANG, Cong-yan LIU, Yan CHEN. Formulation and technology of oxymatrine-astragaloside Ⅳ coloaded liposomes based on quality by design[J]. Acta Pharmaceutica Sinica, 2024 , 59 (1) : 232 -242 . DOI: 10.16438/j.0513-4870.2023-0515

正文

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近年来, 以PD-1、PD-L1抑制剂为代表的免疫检查点抑制剂对某些肿瘤(如肝癌、乳腺癌) 疗效不佳^[1], 这主要与肿瘤复杂的微环境及免疫细胞的功能耗竭有关^[2], 从干预肿瘤相关成纤维细胞(cancer-associated fibroblasts, CAFs) 功能和提高免疫细胞活性的角度出发, 与PD-1抑制剂联用是提高其免疫疗效的策略之一。目前已有研究表明, 中药黄芪中的主要活性成分黄芪甲苷(astragaloside Ⅳ, As) 可通过提高肿瘤浸润性淋巴细胞(tumor infiltrating lymphocytes, TILs) 线粒体活性, 增强瘤内TILs杀伤能力^[3-6]。本课题组前期研究亦发现, 苦参中的主要活性成分氧化苦参碱(oxymatrine, Om) 可通过抑制CAFs活化, 提高瘤内TILs数量^[7], 并且将As与Om按照1∶1~1∶2的比例配伍使用时, 可协同增强PD-1抑制剂的抗肝癌、抗乳腺癌药效。但两药理化性质差异较大, As为难溶性成分, 属于生物药剂学分类系统(biopharmaceutics classification system, BCS) Ⅳ类药物^[8], 而Om为水溶性成分, 属于BCS I类药物, 如何将两药按照最佳配比进行共载和共递送将面临巨大挑战, 也是本研究拟解决的关键药剂学问题。

脂质体是一种由磷脂和胆固醇构成的类脂双分子层闭合囊泡, 具有独特的脂质双分子层和亲水性空腔结构, 可同时包载疏水性药物与亲水性药物。作为商业化程度较高的纳米药物载体^[9], 脂质体制备工艺成熟, 易于工业化生产和临床转化, 是实现Om和As共载与共递送的理想载体, 但目前已上市的两药共载脂质体共载的均为水溶性药物, 尚未见有共载极性差异极大的两种药物的脂质体产品上市, 这可能是由于这种共载脂质体的构建过程涉及多个复杂环节和步骤, 且终产品工艺放大生产难度高、批次间重复性差、质量难以达到预期目标。质量源于设计(quality by design, QbD) 是人用药物注册技术标准国际协调会(International Conference on Harmonisation, ICH) 于2005年提出的药品开发与生产理念^[10], 它强调对药品本身属性及制造过程进行深入研究以确保最终质量^[11], 从而构造科学化、简约化的药品制备工艺体系, 降低药品的开发成本和难度。目前, QbD理念在口服等固体制剂领域的应用已较为成熟, 但在脂质体特别是双药共载脂质体等纳米制剂领域的研究较少。

因此, 本研究基于QbD理念, 以大豆卵磷脂(soybean phosphatidylcholine, SPC) 和氢化大豆卵磷脂(hydrogenated soybean phosphatidylcholine, HSPC) 为脂质材料, 采用乙醇注入法联合pH梯度法构建Om-As共载脂质体(oxymatrine-astragaloside Ⅳ coloaded liposomes, Om-As-Lip), 通过预先设定最终的目标产品质量属性(quality target product profile, QTPP) 和关键质量属性(critical quality attributes, CQAs), 使用双重风险分析工具和Plackett Burman试验筛选出对CQAs影响最显著的关键物料属性(critical material attributes, CMAs), 并运用Box-Behnken设计试验对CMAs参数进行优化, 建立设计空间; 之后进一步对Om-As-Lip的放大工艺进行考察, 优化高压匀质过程的关键工艺参数(critical process parameters, CPPs), 并对终产品进行质量评价, 最终开发出工艺稳定可靠、质量均一可控的Om-As-Lip, 以期为其后续转化应用提供技术参考。

材料与方法

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试剂黄芪甲苷(批号: S31401-1g, 纯度≥ 98%, 上海源叶生物有限公司); 氧化苦参碱(批号: 16837528, 纯度≥ 98%, 南京春秋制药公司); 大豆卵磷脂(批号: 510430-21, 纯度≥ 85%, 德国Lipoid公司); 氢化大豆卵磷脂(批号: B60455, 纯度≥ 95%)、蛋黄卵磷脂(批号: EK14005, 纯度≥ 98%) (上海艾伟拓医药科技有限公司); DSPE-mPEG₂₀₀₀ (批号: R053501, 纯度≥ 98%)、胆固醇(批号: R008056, 纯度≥ 99%) (上海罗恩试剂); 无水碳酸钠(批号: 060410339, 南京化学试剂有限公司); 一水合柠檬酸(批号: 201707052, 国药集团试剂有限公司); N-2-羟乙基哌嗪-N-2-乙磺酸(批号: BS106-500 g, 安徽Biosharp^®公司); 无水乙醇(分析纯, 西陇科学股份有限公司); 乙腈(色谱纯, 美国TEDIA公司)。

仪器 FA2014型万分之一电子天平(上海良平仪器仪表公司); MSZOSDU型十万分之一电子天平(美国梅特勒-托利多仪器有限公司); Agilent Technologies 1260 Series高效液相色谱(美国安捷伦公司); 6100型ELSD蒸发光散射检测器(四川奥泰医疗系统有限责任公司); ZNCL-BS型智能磁力搅拌仪(上海利亚蒙制冷科技有限公司); RE2000A型旋转蒸发仪(上海亚荣生化仪器厂); ST 16R型高速冷冻离心机(美国Thermo Fisher Scientific公司); XO-1000D型超声探头细胞粉碎仪(南京先欧仪器制造有限公司); ZEN 3600型马尔文激光粒度分析仪(英国马尔文仪器有限公司); Tecnai 12型TEM电镜(美国FEI公司); ZRS-8G智能溶出试验仪(天津天光光学仪器有限公司); mAH-1500 N型高压匀质仪(苏州ATS公司)

Om和As的HPLC测定 Om测定条件: 伊利特ODS-2柱(4.6 mm × 250 mm, 5 μm); 流动相为乙腈-水(含0.1%磷酸-0.16%三乙胺) 10∶90, 等度洗脱; 流速0.8 mL·min^-1; 检测波长: 220 nm; 柱温30 ℃; 进样量10 μL。

As测定条件: 伊利特ODS-2柱(4.6 mm × 250 mm, 5 μm); 流动相为乙腈-水35∶65, 等度洗脱; 流速1.0 mL·min^-1; 柱温35 ℃; 进样量10 μL。ELSD漂移管温度110 ℃; 载气流速1.8 L·min^-1; 增益系数1。

Om-As-Lip评价指标的建立

包封率测定取500 μL Om-As-Lip溶液, 经0.22 μm的水系滤膜过滤, 以除去游离的As, 加入2倍量甲醇至续滤液中, 超声加热30 min至完全破乳, 离心取上清液, HPLC检测脂质体中包载的As含量, 计为W₁。之后精密移取500 μL Om-As-Lip滤液置于10 kD超滤离心管中, 13 000 r·min^-1离心15 min, 收集下室离心液, 检测游离的Om含量, 计为W₂。另取500 μL Om-As-Lip溶液, 加入2倍量甲醇, 超声30 min破乳, 分别测定As与Om含量, 记为W₃和W₄。按公式(1、2) 计算包封率(encapsulation efficiency, EE)。

(1)

$ \mathrm{EE}_{\mathrm{Om}}=\left(W_4-W_2\right) / W_4 \times 100 \% $

(2)

$ \mathrm{EE}_{\mathrm{As}}=W_1 / W_3 \times 100 \% $

载药量测定精密吸取1 mL Om-As-Lip溶液, 置于透析袋(MWCO: 10 kD) 中, 流水透析4 h。吸取1 mL透析后溶液, 冷冻干燥后得冻干粉末, 精确称重冻干粉末重量M_总。向冻干物中加入1 mL甲醇涡旋复溶, 13 000 r·min^-1离心10 min, 取上清液分别测定As与Om含量, 记为W₅和W₆。按公式(3、4) 计算载药量(drug loading efficiency, LE)。

(3)

$ \mathrm{LE}_{\mathrm{Om}}=W_6 / M_{\text {总 }} \times 100 \% $

(4)

$ \mathrm{LE}_{\mathrm{As}}=W_5 / M_{\text {总 }} \times 100 \%$

DLS测定取适量的Om-As-Lip溶液, 经超纯水稀释100倍后, 采用马尔文粒径仪对其粒径、PDI和zeta电位进行分析, 所有样品均在25 ℃下测试3次。

Om-As-Lip的制备与处方优化

Om-As-Lip的QTPP与CQAs根据前期研究和文献^[12]综述, 确定本制剂的制剂形式、给药途径、共载比例等, 根据FDA颁布的脂质体药物生产指南及2020年版《中国药典》微粒制剂中对脂质体的相关规定^{[13, 14]}, 在表 1中定义了Om-As-Lip的QTPP和CQAs。

Om-As-Lip的制备分别精密称取适量HSPC、SPC、胆固醇、DSPE-mPEG₂₀₀₀和As, 置于西林瓶中, 加入一定体积的无水乙醇, 超声溶解至澄清透明, 即得油相。在磁力搅拌(800 r·min^-1, 50 ℃) 条件下, 将油相匀速注入5 mL一定浓度的柠檬酸溶液(水相) 中, 搅拌1 h后, 继续加入1 mL HEPES (50 mmol·L^-1) 缓冲盐溶液, 再加入4 mL预溶有Om的碳酸钠溶液(300 mmol·L^-1), 调节pH值为7.0, 50 ℃水合孵育一定时间, 加超纯水定容至10 mL, 即得Om-As-Lip。

风险评估采用鱼骨图分析影响CQAs的风险因素。将FEMA作为第二工具从失效影响、潜在原因、控制方法等角度综合分析, 结合风险指数[RPN, 即严重程度(S)、发生率(O) 和检测率(D) 3种指标的乘积] 对各个风险因素进行评级。参照FDA脂质体生产指南^[14]和ICH药品研发指南Q9^[10]制定S、O和D的评判标准, 从1 (影响最低) ~5 (影响最高) 进行评分, 按公式(5) 进行计算:

(5)

${\rm RPN = S × O × D} $

评判标准: RPN > 16或S = 4时为高风险, 16 ≥ RPN ≥ 8时为中风险, RPN ≤ 7时为低风险。

Plackett-Burman设计筛选关键影响因素在前期实验基础上, 结合对Om-As-Lip处方和制备工艺的风险分析结果, 以As药脂比(X₁)、Om药脂比(X₂)、磷脂浓度(X₃)、胆脂比(X₄)、DSPE-mPEG₂₀₀₀浓度(X₅)、HSPC与SPC质量比(X₆)、无水乙醇体积(X₇)、pH梯度(X₈)、孵育时间(X₉) 为考察对象, 以Om与As两药总包封率(Y₁) 为评价指标, 利用Plackett-Burman实验设计筛选出对Om-As-Lip性质具有显著影响的因素。

Box-Behnken响应面设计优选CMAs配比在Plackett-Burman实验的基础上, 以As包封率(Y₁)、Om包封率(Y₂) 为响应值, 选取As药脂比(X₁)、胆脂比(X₂)、HSPC∶SPC质量比(X₃) 为响应面模型自变量, 其他因素固定为: Om投药量为2.50 mg, 磷脂浓度为0.60 mg·mL^-1, DSPE-mPEG₂₀₀₀投药量为2.00 mg、无水乙醇体积为2.0 mL、pH梯度为5.0、孵育时间为3.0 h, 总体积定容至10.0 mL, 进行三因素三水平的Box-Behnken实验设计, 确定最佳工艺处方。

设计空间的建立根据响应面实验结果, 固定As药脂比为1∶40, 根据胆脂比和混合磷脂比例对Om和As包封率影响的等值曲线进行叠加, 建立设计空间, 为后续工艺放大确定最优工艺条件范围。由于在放大生产的匀质环节中, 脂质体会受到匀质机的剪切、空化冲击, 导致药物包封率在一定程度上有所降低^[15], 因此本研究将As包封率下限调整为80%, Om包封率下限调整为50%。以此为条件搜索符合目标的子集, 构建设计空间。

Om-As-Lip放大工艺的CPPs研究 在实验室小试的基础上, 将基于QbD理念优化的Om-As-Lip处方放大50倍得到50×-Om-As-Lip, 检测其CQAs是否合格。采用单因素实验对50×-Om-As-Lip进行高压匀质(high-pressure homogenization, HPH) CPPs的筛选, 分别考察匀质压力(20、40、60、80、90 bar)、匀质循环次数(3、6、9、12、15次)、匀质温度(4、15、25、35 ℃) 对脂质体CQAs的影响。基于单因素实验结果筛选出高压匀质的最佳CPPs条件, 制备3批放大样品进行高压匀质, 得到HPH-Om-As-Lip, 测定其包封率、粒径、PDI和zeta电位, 验证放大工艺的可重复性。

HPH-Om-As-Lip的质量评价

形态学观察将HPH-Om-As-Lip溶液稀释适宜倍数后, 用2%磷钨酸负染后转移至专用铜网上, 自然晾干, 于透射电镜下观察其双分子层膜结构和大小并拍照。

稳定性考察稀释稳定性: 取HPH-Om-As-Lip 1 mL, 分别用超纯水稀释10、20、50、100倍体积, 测定其粒径和PDI。

放置稳定性: 取HPH-Om-As-Lip 10 mL, 于4 ℃条件下保存, 分别于1、2、3、4、5、6、7、8、14、21、28天定时取样测定其粒径和PDI。

体外释放考察精密吸取5 mL HPH-Om-As-Lip溶液放于透析袋(10 kDa) 中, 将两端用透析夹扎紧, 另取游离药物组合(Om-As) 溶液置于透析袋中, 再将透析袋放置于盛有150 mL含0.5% Tween 80的磷酸盐缓冲液(pH = 7.4) 的溶出杯中, 在(37 ± 0.5) ℃、100 r·min^-1条件下不断搅拌, 分别在0、0.25、0.5、1、1.5、2、4、6、8、10、12、24、36、48 h时吸取1 mL释放介质并补充同体积的释放介质。将取出的样品溶液于4 ℃、13 000 r·min^-1下离心10 min, 取上清液过滤, 采用HPLC法检测Om及As含量。按照公式(6) 计算药物的累计释放百分率(%)。

(6)

$E_{\mathrm{r}}=\frac{V_{\mathrm{e}} \sum\nolimits_1^{n-1} C_{\mathrm{i}}+V_0 C_n}{m_{\text {drug }}} \times 100 \%$

E_r: 药物累计释放率; V_e: PBS的置换体积; V₀: 释放介质总体积; C_i: 第i次置换取样时释放液的浓度; m_drug: 脂质体所含药物总药量; n: 置换PBS的次数。

结果

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1 风险评估

图 1从人员及环境、物料属性、乙醇注入法、pH梯度法和高压匀质的工艺参数5个方面分析了可能影响Om-As-Lip CQAs的风险因素。表 2结果显示, 磷脂浓度、As药脂比、Om药脂比、HSPC与SPC质量比(HSPC∶SPC)、pH梯度、PEG修饰材料(DSPE-mPEG₂₀₀₀) 浓度、无水乙醇体积为影响Om-As-Lip CQAs的中风险或高风险因素。

2 Om-As-Lip处方优化

2.1 Plackett-Burman设计筛选关键影响因素

由于影响双载脂质体CQAs的风险因素较多, 鱼骨图和FEMA作为风险评估工具仍具有一定程度上的主观影响, 因此进一步采用Plackett-Burman实验对影响因素进行分析。包封率是脂质体的关键质量属性, 根据脂质体中药物包封程度的高低, 可评价其处方工艺的优劣。Plackett-Burman实验的主要目的在于对影响脂质体的关键质量属性的关键因素进行排序筛选, 因此本实验将两药包封率之和(Y₁) 作为指标进行考察。Plackett-Burman的因素水平、实验安排及结果见表 3。回归模型为: Y₁ (%) = -17.59X₁ - 0.585 8X₂ + 6.83X₃ - 11.58X₄ + 2.12X₅ - 13.54X₆ + 3.06X₇ + 7.49X₈ - 6.69X₉, R² = 0.991 5, R_adj² = 0.953 3。由图 2A可知, As药脂比、HSPC与SPC质量比(HSPC∶SPC)、胆脂比对两药包封率之和有显著性消极性影响(P < 0.05), 影响大小排序为As药脂比 > HSPC∶SPC > 胆脂比。

2.2 Box-Behnken响应面实验设计优选处方配比

2.2.1 模型拟合及方差分析

表 4为Box-Behnken响应面的因素水平、实验安排及结果。通过Design Expert 13.0软件对表 4数据进行拟合, 建立的二次多项式回归模型方程为Y₁ = 93.64 - 15.26X₁ + 0.618 8X₂ - 0.491 3X₃ + 2.16X₁X₂ - 1.78X₁X₃ - 5.51X₂X₃ - 15.27X₁² - 3.98X₂² + 1.82X₃², Y₂ = 36.58 - 6.05X₁ + 9.83X₂ + 2.01X₃ - 9.97X₁X₂ - 3.87X₁X₃ + 0.83X₂X₃ + 0.435 5X₁² + 1.32X₂² + 4.48X₃²。两个模型P < 0.05, 均具有显著性影响, 而失拟项P > 0.05, 表明失拟均不显著。模型的R²分别为0.940 3、0.909 7, R_adj²分别为0.863 5、0.793 6, 表明模型与实际实验拟合程度良好, 适用于分析和预测Om-As-Lip的制备。

2.2.2 响应面分析

As包封率(Y₁) 的等高线和响应面分析见图 2B、C, 根据等高线的密度分析, 等高线越密集, 影响程度越大。因此, 确定各因素的影响大小顺序为As药脂比(X₁) > HSPC∶SPC (X₃) > 胆脂比(X₂), 其中As具有显著性影响(P < 0.001)。根据响应面图可知, 随着As的药脂比增大, SPC比例减少, 胆脂比增多, As的包封率逐渐降低。Om包封率(Y₂) 的等高线和响应面分析见图 2D、E, 各因素的影响大小顺序为胆脂比 > As药脂比 > HSPC∶SPC, 其中胆脂比(P < 0.001)、As药脂比(P < 0.05) 具有显著性影响, 且两者交互具有显著性(P < 0.05), 随着胆脂比增大, As药脂比降低, SPC质量减少, Om的包封率逐渐升高。

2.2.3 最佳工艺的确定和验证

期望以As和Om的包封率处于最大值为优化指标, 通过Design Expert 13.0软件分析, 最终确定Om-As-Lip制备的最佳CMAs参数为: As药脂比为1∶40, 胆脂比为1∶6, HSPC∶SPC为51∶9。在该条件下重复制备3批Om-As-Lip, 测得As包封率为(97.28 ± 0.01)%、As载药量为(2.81 ± 0.01)%, Om包封率为(60.76 ± 0.01)%、Om载药量为(3.01 ± 0.01)%, RSD < 3%, 两药最终载药比例在1∶1~1∶2之间, 符合预期CQAs。

2.3 设计空间的建立

虽然确定了Om-As-Lip CMAs的最佳取值, 但是放大生产时难以精准控制条件, 导致制备的Om-As-Lip批次重复性差。合理的设计空间可从多维角度考虑工艺参数之间的组合和相互作用, 在风险分析及实验基础上界定出CMAs的一定范围, 在此范围内CMAs的更改将不会改变最终药品的CQAs。为了获取更大的设计空间, 本实验将对包封率影响最显著的As药脂比固定在1∶40, 将胆脂比、HSPC∶SPC对Om包封率和As包封率影响的等值曲线进行叠加, 得出图 2F中黄色区域为建立的设计空间, 但由于模型不可完全预测真实值, 因此加入置信水平α = 0.05的置信区间优化原设计空间(图 2G), 灰色区域中的取值将不满足优化目标, 暗黄色区域为原设计空间中不可靠的部分, 预计此空间中的取值有5%概率无法满足目标, 而亮黄色则为优化后的设计空间, 该区域的胆脂比可控制在1∶12~1∶5, HSPC∶SPC比例可控制在1∶7~17∶3, 此范围内所有取点都将符合CQAs。

2.4 Om-As-Lip放大工艺

将设计空间内的CMAs参数放大50倍进行放大工艺研究, 由于放大后参数取值超出设计空间规定的范围, 制备的50×-Om-As-Lip极有可能不符合CQAs, 因此考虑加入HPH解决这一问题。图 3A、B分别展示了高压匀质3个CPPs (压力、循环次数、温度) 在不同水平下制备的HPH-Om-As-Lip的粒径、PDI和包封率变化, 发现当匀质压力为600 bar时, HPH-Om-As-Lip粒径为(115.1 ± 2.479) nm, PDI为0.216 ± 0.01, Om和As的包封率均最高, 其他压力水平下制备的脂质体的粒径均偏小, 导致形成的内水相空腔也随之减小, 继而影响亲水性药物Om的包封。当高压匀质循环次数为6次时, HPH-Om-As-Lip的粒径为(118.6 ± 1.68) nm, PDI为0.173 ± 0.015, 对Om和As的包封率均为最高, 分别为(50.41 ± 0.02)%和(88.60 ± 0.14)%。当高压匀质温度为4 ℃时, HPH-Om-As-Lip对Om和As的包封率均最高[(49.38 ± 0.09)%, (91.99 ± 0.05)%], 之后随温度上升, 两药包封率均开始降低, 且脂质体粒径开始减小、PDI逐渐升高。根据CQAs规定, HPH-Om-As-Lip的粒径应在100~200 nm之间, PDI < 0.3, 且具有高的包封率, 综合考虑不同高压匀质因素水平对HPH-Om-As-Lip包封率、粒径、PDI的影响后, 选择匀质压力为600 bar、循环次数为6次、匀质温度为4 ℃作为高压匀质最优的CPPs。

在上述优化的参数条件下, 重复制备3批HPH-Om-As-Lip进行优化工艺验证, 3批样品溶液外观均澄清透亮, 粒径为(116.4 ± 5.2) nm, PDI为0.196 ± 0.028, zeta电位为(-32.5 ± 1.19) mV。Om包封率为(55.35 ± 1.38)%, Om载药量为(2.271 ± 0.001)%, As包封率为(91.21 ± 0.27)%, As载药量为(2.293 ± 0.001)%, RSD < 3%, 由此可见, 此放大工艺具有良好的稳定性。

2.5 HPH-Om-As-Lip的质量评价

如图 3C所示, HPH-Om-As-Lip的溶液外观无色澄清, 有蓝色乳光, 与实验室小试样品基本一致, DLS结果显示粒径为(115.1 ± 2.5) nm, PDI为0.22 ± 0.01, zeta电位为(-36.6 ± 1.7) mV。图 3D中的TEM电镜结果显示, 大部分HPH-Om-As-Lip形态圆整, 呈类球形, 可观察到明显的脂质双分子层结构(白色箭头)。而未经高压匀质工艺制备的50×-Om-As-Lip的溶液外观相较于小试样品Om-As-Lip而言变得较为浑浊, 其粒径为(148.3 ± 28.4) nm, PDI为0.704 ± 0.227, DLS粒径表征图出现多个峰, 与图 3D TEM结果一致, 其中10 nm左右的峰可能来源于磷脂碎片(TEM图中蓝色箭头所指), 100~200 nm之间的峰应为目标制剂(TEM图中白色箭头所指), 1 000 nm左右的峰表示脂质可能发生了聚集(TEM图中黄色箭头所指)。该现象表明, 脂质体作为微观制剂, 放大工艺对其CQAs影响极大, 实验室小试规模下建立的设计空间可能并不完全适用于放大工艺。放大处方制备的50×-Om-As-Lip的粒径分布不均一, 可能是由于实验室的超声及磁力搅拌提供的能量不够, 因此高压匀质是Om-As-Lip放大工艺必不可少的环节。

各组的体外释放结果如图 3E所示, 游离药物组合(Om-As) 中的Om在1.5 h时的累计释放率超过80%, 几乎完全释放; 而HPH-Om-As-Lip组中的Om累计释放率约为55%; 游离药物组合的As在8 h时累计释放率超过80%, 而HPH-Om-As-Lip组的累计释放率只有20%。与游离药物组合相比, HPH-Om-As-Lip组表现出明显的缓释特性。采用零级方程、一级方程、Higuchi方程、Riger-Peppas方程4种释药模型方程对HPH-Om-As-Lip的释药曲线进行拟合, 结果发现一级方程的拟合效果最好, 其中Om的拟合方程为Q_t = 87.24 [1-exp (-0.85t)], As的拟合方程为Q_t = 0.54 [1-exp (-0.08t)], R²分别为0.947 9和0.915 0, 拟合效果较好。2020版药典规定缓释制剂的释放动力学方程应符合一级方程或Higuchi方程, 因此HPH-Om-As-Lip的体外释放符合药典对缓释制剂的规定要求。

对HPH-Om-As-Lip的稀释稳定性和放置稳定性进行考察, 结果如图 3F所示, 在不同稀释倍数下, HPH-Om-As-Lip的粒径、PDI均未发生明显变化; HPH-Om-As-Lip在考察的放置时间内均可保持其粒径稳定在111.5 nm左右, PDI值均低于0.3, 表明HPH-Om-As-Lip具有较好的物理稳定性。

讨论

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目前已有研究发现, 免疫疗法的疗效与TILs的瘤内浸润数量和质量密切相关^[16]。本课题组前期研究发现, 将As和Om按1∶1~1∶2的比例联合使用时, 可协同增强T细胞活性, 并抑制CAFs活化, 进而显著提高PD-1抑制剂的抗肝癌、抗乳腺癌药效。但As与Om理化性质差异极大, 将两药按固定比例实现共载与体内共递送存在极大挑战。基于药物的理化性质和脂质体的载药特点, 本研究拟构建一种载药量高、性质稳定、可放大生产的双药共载脂质体(Om-As-Lip), 即将难溶性As负载于脂质体的脂质双分子层中, 水溶性Om负载于内水相中, 并在QbD理念的指导下, 建立稳定可控的处方工艺体系, 以期为肿瘤免疫治疗提供疗效确切、安全有效的辅助治疗药物。

虽然脂质体的制备工艺已经较为成熟, 但目前多数脂质体药物研发仍处于实验室小试水平, 放大生产仍存在诸多困难^{[17, 18]}。目前全球已获批上市的脂质体产品约有20种, 且多为单药脂质体, 共载脂质体相对较少。其中由Celator公司研发生产的共载阿糖胞苷和柔红霉素的Vyxeos^®于2016年被批准用于治疗白血病, 最近国内研制的首个两药共载复方脂质体-盐酸伊立替康氟脲苷脂质体注射液(LY01616) 已于2021年完成I期临床试验。但上述脂质体产品共载的药物均为水溶性药物, 对于共载极性完全不同的两种小分子药物的Om-As-Lip而言, 目前尚未有充足的研发经验可作为参考。

作为脂质体的主要组成成分, 磷脂种类是影响脂质体CQAs的关键因素之一。磷脂种类可分为天然磷脂和合成磷脂, 其中常用于脂质体制备的天然磷脂包括蛋黄卵磷脂(egg hosphatidylcholine, EPC) 和SPC, 这两种磷脂对疏水性药物均有较好的增溶效果, 这可能是由于天然磷脂含有多种不同碳链长度的脂肪酸链结构^[19], 更易于结合疏水性药物。以HSPC为代表的合成磷脂制备的脂质体对水溶性药物通常具有更优的包封效率, 这可能是由于合成磷脂多具有较高的相变温度(phase transformation temperature, T_m), 导致脂质双分子层更稳定, 不易发生药物泄漏, 其次是因为合成磷脂多具有较长的烃链^{[20, 21]}, 可形成更大的内水腔, 进而有利于负载更多的水溶性药物, 但同时制备的脂质体粒径也会随之增大。本研究前期考察了不同种类的磷脂对As和Om的包封效率, 结果发现SPC对As的包封率高达98.04%, 显著高于EPC (78.37%) 和HSPC (58.29%), 而HSPC对Om具有更优的包封率(71.13%), 显著高于EPC (19.32%), SPC (9.67%)。实验结果还发现, 随磷脂的碳链长度增加, 脂质体的粒径也在增加(EPC : 147.8 nm, SPC: 160.0 nm, HSPC: 216.2 nm)。因此, 综合考虑不同种类磷脂的载药特点和拟包载药物的理化性质, 本研究将SPC与HSPC两种磷脂混合使用作为Om-As-Lip的脂质材料。

脂质体的制备方法也是影响其CQAs的关键因素。根据载药机制可将脂质体的制备方法大致分为主动载药法和被动载药法。其中, 主动载药法常用于包载水溶性药物, pH梯度法是一种经典的主动载药法, 其主要原理是通过调节脂质体内外水相的pH梯度差, 使药物以易跨膜的分子态顺梯度到达内水腔后, 转变为不易跨膜的离子态被包封。经典的被动载药法如乙醇注入法、薄膜分散法适合包载疏水性药物。乙醇注入法的原理是将溶有脂质的乙醇溶液注入到水相中, 使得脂质在乙醇-水界面中迅速分散形成脂质体^[22], 形成的脂质体粒径较小, 操作简单、易于工业化生产^{[23, 24]}。薄膜分散法是将药物和脂质溶于易挥发的有机溶剂(如氯仿) 后, 除去有机溶剂形成一层薄膜, 再加入水化溶液形成脂质体。本研究前期在对制备方法进行筛选时, 发现已有大量研究证明采用pH梯度法制备的单药脂质体对Om具有更高的包封率(pH梯度法约为50%~60%, 被动载药法低于30%)^{[7, 25, 26]}, 因此考虑采用pH梯度法将Om加载于脂质体内水腔, 同时采用被动载药法将As包封于脂质双分子层间。实验前期分别对乙醇注入法-pH梯度法和薄膜分散法-pH梯度法制备的脂质体进行了比较, 结果发现采用乙醇注入法-pH梯度法制备的脂质体对As的包封率(84.54%) 显著高于薄膜分散法-pH梯度法(49.82%), 主要原因在于As在氯仿中基本不溶, 形成的薄膜不均匀; 两种制备方法制备的脂质体对Om的包封率未见有显著差别(34.97%和39.26%)。本研究也曾参考Kuo等^[27]的做法, 将As预先溶于甲醇, 再与氯仿混合, 但发现该法在除去有机溶剂环节耗费的时间较长, 不利于工业化生产, 因此, 本研究最终选择采用乙醇注入法-pH梯度法制备Om-As-Lip。

在脂质体的放大工艺中, 常面临粒径不均一、批次间重复性差的难题。高压匀质可在高压条件下将脂质体样品迅速通过具有特殊结构的匀质腔, 通过产生剪切、撞击、空穴等效应, 从而降低脂质体的粒径及PDI, 有助于克服粒径不均一的难题, 促进脂质体进行产业转化。高压匀质的压力、循环次数均有利于降低脂质体的粒径和PDI, 但由于脂质体刚性不强, 压力过高、循环次数过多则可能破坏脂质体结构, 降低药物的包封率。同时, 在高压匀质机工作时产生的热量会使T_m较低的磷脂通透性增加, 进而导致药物包封率降低, 因此需要将匀质温度控制在较低范围。因此, 本实验最终确定匀质压力为600 bar, 匀质次数为6次, 匀质温度为4 ℃作为最优高压匀质工艺参数, 在此条件下放大50倍生产的HPH-Om-As-Lip粒径均一、包封率和载药量与小试相比并未有明显下降, 产品质量符合CQAs。

综上所述, 脂质体的CMAs和CPPs均对其CQAs有较大的影响, 深刻理解它们之间的联系将有助于简化制剂设计, 降低生产难度, 推进临床转化^[28]。QbD理念是优化制剂处方工艺、获得质量稳定可控产品的重要技术方法, 虽然本研究中Om-As-Lip的放大研究规模距离真正的产业化仍有一定差异，但在QbD理念下建立的Om-As-Lip制备工艺体系稳定可放大, 产品质量均一可控, 且成功实现了不同极性药物的高效共载(EE-As: 97.28%, EE-Om: 60.76%), 在放大后仍保持较高的包封率(EE-As: 91.21%, EE-Om: 55.35%), 因此Om-As-Lip仍具有一定应用前景。该研究有望为类似脂质体制剂的研究开发提供技术参考, 也可为肿瘤免疫治疗提供更有效的辅助治疗药物。

作者贡献: 韦良银负责全部实验内容与结果处理, 并负责文章撰写与修改; 李霞、王虹、黄琳清协助Om-As-Lip制备优化及提供思路; 刘聪燕负责文章修改; 陈彦负责实验方案设计、实验指导与结果审核、文章修改与审核。

利益冲突: 所有作者均声明不存在利益冲突。

基金

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国家自然基金资助项目(82173985)
江苏省重点研发计划社会发展项目(BE2021754)

参考文献

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2024年第59卷第1期

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doi: 10.16438/j.0513-4870.2023-0515

接收时间：2023-04-27
首发时间：2025-11-28
出版时间：2024-01-12

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收稿日期：2023-04-27
修回日期：2023-09-05

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国家自然基金资助项目(82173985)

江苏省重点研发计划社会发展项目(BE2021754)

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1.南京中医药大学附属中西医结合医院, 江苏南京 210028

2.江苏省中医药研究院, 中药组分与微生态研究中心, 江苏南京 210028

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^*刘聪燕，Tel: 86-25-52362155, E-mail: liucongyan2007@126.com;

陈彦, E-mail: ychen2020@hotmail.com

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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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Element	Quality attribute	Target	Justification
QTPP	Route of administration	Intravenous	Increase the bioavailability of active substances at the tumor site
Dosage form	Liposome	Co-encapsulate and co-delivery of Om and As
Outer packing	Penicillin bottle	Ensure stability of Om-As-Lip during storage
Prolonged blood circulation time	PEGylated liposomes	To avoid reticuloendothelial system macrophages
Appearance	Clear and transparent	Quality standards for injections
In vitro release	Slow release	Standards for sustained-release preparations
CQAs	EE%	50%-100%	Reduced drug loss and production costs
Size	100-200 nm	Produce EPR effect and enhance drug concentration
PDI	< 0.3	To predict the behavior of Om-As-Lip in vivo
Zeta potential	≤ -20 mV / ≥ 20 mV	To enhance the stability
As∶Om	1∶1-1∶2	To achieve the desired effect at the tumor site

Element

Quality attribute

Target

Justification

QTPP

Route of administration

Intravenous

Increase the bioavailability of active substances at the tumor site

Dosage form

Liposome

Co-encapsulate and co-delivery of Om and As

Outer packing

Penicillin bottle

Ensure stability of Om-As-Lip during storage

Prolonged blood circulation time

PEGylated liposomes

To avoid reticuloendothelial system macrophages

Appearance

Clear and transparent

Quality standards for injections

In vitro release

Slow release

Standards for sustained-release preparations

CQAs

EE%

50%-100%

Reduced drug loss and production costs

Size

100-200 nm

Produce EPR effect and enhance drug concentration

PDI

< 0.3

To predict the behavior of Om-As-Lip in vivo

Zeta potential

≤ -20 mV / ≥ 20 mV

To enhance the stability

As∶Om

1∶1-1∶2

To achieve the desired effect at the tumor site

Parameter	Failure mode	Failure effect	Potential cause	Control method	S	D	O	RPN
EE/%	Drug leakage	Increase administration frequency	Phospholipids concentration	Confirm the appropriate concentration range	4	3	4	48
As concentration	4	3	4	48
Om concentration	4	3	3	48
Cholesterol concentration	4	3	3	36
Synthetic phospholipids ratio	Optimizing the phospholipid ratio	4	3	4	48
Om loading method	pH gradient	3	1	2	6
The pH of the hydration medium	Optimizing the citric acid concentration	4	2	4	32
Incubation time	Optimizing incubation time	4	2	3	24
Size, PDI	Large size distribution	Reduced stability	Uniform particle size	Magnetic stirring speed	2	1	3	6
Complex process in vivo	Ultrasound time	2	1	3	6
Zeta potential	> -20 mV	Aggregation during storage life	Types of phospholipids	Use highly charged phospholipid	2	1	5	10
Phospholipids concentration	Optimize phospholipids concentration	4	3	4	48
Prolong blood circulation time	Eliminated by the RES system	Reduced effectiveness of treatment	PEG modified liposomes	Optimize DSPE-mPEG₂₀₀₀ concentration	3	2	3	18
Increased side effects
Safety	Residual organic solvent	Increased side effects	Volume of absolute ethanol	Optimize the volume of absolute ethanol	4	3	3	36
Reduced storage stability

Parameter

Failure mode

Failure effect

Potential cause

Control method

RPN

EE/%

Drug leakage

Increase administration frequency

Phospholipids concentration

Confirm the appropriate concentration range

As concentration

Om concentration

Cholesterol concentration

Synthetic phospholipids ratio

Optimizing the phospholipid ratio

Om loading method

pH gradient

The pH of the hydration medium

Optimizing the citric acid concentration

Incubation time

Optimizing incubation time

Size, PDI

Large size distribution

Reduced stability

Uniform particle size

Magnetic stirring speed

Complex process in vivo

Ultrasound time

Zeta potential

> -20 mV

Aggregation during storage life

Types of phospholipids

Use highly charged phospholipid

Phospholipids concentration

Optimize phospholipids concentration

Prolong blood circulation time

Eliminated by the RES system

Reduced effectiveness of treatment

PEG modified liposomes

Optimize DSPE-mPEG₂₀₀₀ concentration

Increased side effects

Safety

Residual organic solvent

Increased side effects

Volume of absolute ethanol

Optimize the volume of absolute ethanol

Reduced storage stability

Run	X₁	X₂	X₃/mg·mL^-1	X₄	X₅	X₆	X₇/mL	X₈	X₉/h	Y₁/%
PB-1	1 (-1)	3 (1)	0.6 (1)	12 (1)	1 (-1)	11∶1 (-1)	2 (-1)	5 (1)	0.5 (-1)	156.94
PB-2	1 (-1)	1 (-1)	0.6 (1)	3 (-1)	4 (1)	7∶1 (1)	2 (-1)	5 (1)	3 (1)	140.90
PB-3	3 (1)	1 (-1)	0.6 (1)	12 (1)	4 (1)	11∶1 (-1)	2 (-1)	2 (-1)	3 (1)	101.94
PB-4	3 (1)	1 (-1)	0.3 (-1)	3 (-1)	4 (1)	11∶1 (-1)	5 (1)	5 (1)	0.5 (-1)	142.80
PB-5	1 (-1)	1 (-1)	0.3 (-1)	3 (-1)	1 (-1)	11∶1 (-1)	2 (-1)	2 (-1)	0.5 (-1)	151.59
PB-6	1 (-1)	1 (-1)	0.3 (-1)	12 (1)	1 (-1)	7∶1 (1)	5 (1)	2 (-1)	3 (1)	95.11
PB-7	1 (-1)	3 (1)	0.3 (-1)	12 (1)	4 (1)	11∶1 (-1)	5 (1)	5 (1)	3 (1)	139.22
PB-8	3 (1)	1 (-1)	0.6 (1)	12 (1)	1 (-1)	7∶1 (1)	5 (1)	5 (1)	0.5 (-1)	100.94
PB-9	3 (1)	3 (1)	0.3 (-1)	12 (1)	4 (1)	7∶1 (1)	2 (-1)	2 (-1)	0.5 (-1)	66.13
PB-10	3 (1)	3 (1)	0.6 (1)	3 (-1)	1 (-1)	11∶1 (-1)	5 (1)	2 (-1)	3 (1)	118.52
PB-11	1 (-1)	3 (1)	0.6 (1)	3 (-1)	4 (1)	7∶1 (1)	5 (1)	2 (-1)	0.5 (-1)	151.52
PB-12	3 (1)	3 (1)	0.3 (-1)	3 (-1)	1 (-1)	7∶1 (1)	2 (-1)	5 (1)	3 (1)	93.92

Run

X₁

X₂

X₃/mg·mL^-1

X₄

X₅

X₆

X₇/mL

X₈

X₉/h

Y₁/%

PB-1

1 (-1)

3 (1)

0.6 (1)

12 (1)

1 (-1)

11∶1 (-1)

2 (-1)

5 (1)

0.5 (-1)

156.94

PB-2

1 (-1)

0.6 (1)

3 (-1)

4 (1)

7∶1 (1)

2 (-1)

5 (1)

3 (1)

140.90

PB-3

3 (1)

1 (-1)

0.6 (1)

12 (1)

4 (1)

11∶1 (-1)

2 (-1)

3 (1)

101.94

PB-4

3 (1)

1 (-1)

0.3 (-1)

3 (-1)

4 (1)

11∶1 (-1)

5 (1)

0.5 (-1)

142.80

PB-5

1 (-1)

0.3 (-1)

3 (-1)

1 (-1)

11∶1 (-1)

2 (-1)

0.5 (-1)

151.59

PB-6

1 (-1)

0.3 (-1)

12 (1)

1 (-1)

7∶1 (1)

5 (1)

2 (-1)

3 (1)

95.11

PB-7

1 (-1)

3 (1)

0.3 (-1)

12 (1)

4 (1)

11∶1 (-1)

5 (1)

3 (1)

139.22

PB-8

3 (1)

1 (-1)

0.6 (1)

12 (1)

1 (-1)

7∶1 (1)

5 (1)

0.5 (-1)

100.94

PB-9

3 (1)

0.3 (-1)

12 (1)

4 (1)

7∶1 (1)

2 (-1)

0.5 (-1)

66.13

PB-10

3 (1)

0.6 (1)

3 (-1)

1 (-1)

11∶1 (-1)

5 (1)

2 (-1)

3 (1)

118.52

PB-11

1 (-1)

3 (1)

0.6 (1)

3 (-1)

4 (1)

7∶1 (1)

5 (1)

2 (-1)

0.5 (-1)

151.52

PB-12

3 (1)

0.3 (-1)

3 (-1)

1 (-1)

7∶1 (1)

2 (-1)

5 (1)

3 (1)

93.92

Run	Level		Factor		Result
1	-1	0	1		1	10	51∶9		93.13	49.72
2	0	0	0	2	10	52.5∶7.5	89.87	42.34
3	1	0	1	3	10	51∶9	62.88	31.31
4	0	-1	-1	2	8	54∶6	80.57	27.28
5	1	-1	0	3	8	52.5∶7.5	58.02	32.82
6	1	0	-1	3	10	54∶6	70.80	41.01
7	0	0	0	2	10	52.5∶7.5	98.49	35.91
8	-1	1	0	1	12	52.5∶7.5	86.43	63.79
9	0	1	1	2	12	51∶9	91.38	59.14
10	-1	-1	0	1	8	52.5∶7.5	96.69	26.40
11	0	-1	1	2	8	51∶9	93.99	35.63
12	0	0	0	2	10	52.5∶7.5	94.52	39.78
13	0	0	0	2	10	52.5∶7.5	93.53	31.85
14	1	1	0	3	12	52.5∶7.5	56.41	30.33
15	-1	0	1	1	10	51∶9	93.94	43.96
16	0	1	1	2	12	51∶9	100.0	47.47
17	0	0	0	2	10	52.5∶7.5	91.78	33.04

Run

Level

Factor

Result

X₁

X₂

X₃

X₁

X₂

X₃

Y₁/%

Y₂/%

-1

51∶9

93.13

49.72

52.5∶7.5

89.87

42.34

51∶9

62.88

31.31

-1

54∶6

80.57

27.28

-1

52.5∶7.5

58.02

32.82

-1

54∶6

70.80

41.01

52.5∶7.5

98.49

35.91

-1

52.5∶7.5

86.43

63.79

51∶9

91.38

59.14

-1

52.5∶7.5

96.69

26.40

-1

51∶9

93.99

35.63

52.5∶7.5

94.52

39.78

52.5∶7.5

93.53

31.85

52.5∶7.5

56.41

30.33

-1

51∶9

93.94

43.96

51∶9

100.0

47.47

52.5∶7.5

91.78

33.04