Freeze-drying protective agent on the active components of HB liposome encapsulation efficiency and particle size of
Author: Juan Hu Shui-Gen BIAN Jun Wang Guohua
Ke heaven and earth
【Abstract】 Objective To study the different kinds and
concentrations of freeze-dried protective agent against HB
before and after the effective components of freeze-dried
liposome encapsulation rate and the average particle size effects. Method prescribed by 16 active components of HB was
prepared liposomes, using fluorescence spectrophotometry encapsulation rate, with a microscope and digital image analysis software determined the average particle size, before and after the adoption of freeze-dried liposome encapsulation
rate and Comparison of the optimized size of the freeze-drying
protective agent. The results of the best protective agent for the freeze-dried mannitol and binary protective agent sugar -
glycine, the encapsulation rate of 69.91% and 66.70%, appearance good effect, the ratio, respectively, mannitol: PC: CH: HB = 4:1 : 0.5:0.2; sucrose: glycine: PC: CH: HB = 4:0.8:1:0.5:0.2. Conclusion protective agent can reduce the freeze-dried liposome vesicle at the freeze-drying process of
the damage as well as liposome encapsulation efficiency and
particle size effects.
Key words liposome entrapment efficiency of freeze-dried
protective agent particle size
Abstract: ObjectiveTo study the effects of freeze-drying
on the endcapsulation and vesicles size of HB liposome with different kinds of lyoprotectents at various
concentrations.MethodsSixteen formulations of HB liposome were designed.The entrapment efficiency was examined by
fluorospectrophotometry.The particle size of the preparation was examined by microscope and digital imaging analysis
software.The optimized lyoprotectent was selected based on the comparison of entrapment efficiency and particle size before and after freeze-drying.ResultsThe use of sucrose as a primary lyoprotectent and glycine as a supporting lyoprotectents and
the use of mannitol provided the highest encapsulation rate and minimal change of HB liposomes.The results showed that the optimum formula were Man: PC: CH: HB = 4:1:0.5:0.2; Suc: Gly: PC: CH: HB = 4:0.8 : 1:0.5:0.2, the encapsulation rates were
69.91% and 66.70%. ConclusionThe lyoprotectent can protect the liposome vesicles during freeze-drying and mininal the
influences of encapsulation and particle size.
Key words: Liposome; Lyoprotectents; Entrapment efficiency; Particle size
HB effective components from the shells of marine molluscs Park Limnology class Anodonta mussels anodonta woodiana (Lea) extracted with a group of anti-tumor activity
of glycoproteins substances. Made of the effective components of the liposomes by transplanted tumors in mice in vivo
indicate that oral administration with anti-tumor activity
against S180 sarcoma, Lewis lung cancer, lung metastasis by B16 melanoma tumor inhibition rates were 48.76%, 54.38%, 52.66% . However, active components of liquid HB-miscible
emulsion of liposomes is only stored a few weeks , at 50 ?
or more media environment, the structure is unstable, phosphate ester bilayer structure is likely to oxidative damage, not only affects the drug encapsulation efficiency, and the resulting lysophosphatidic would endanger the body . In order to liposome long-term stability at room
temperature storage, widely used in recent years, freeze-dried
products prepared by freeze-drying technique. Preparation of
freeze-drying method is the best way to store liposomes.
However, the process of the formation of ice crystals in the freezer so that liposome fusion gathered in the freezing and thawing process, the formation of ice crystals inside and outside the membrane caused by osmotic pressure difference at
different rates, resulting in liposome lysis, so freeze-drying
process, should be added cryoprotectants (CAP) in order to reduce damage. This method solved the liposomes prepared by
the process and storage of the physical stability and seepage
problems, and avoid the instability of drug inactivation and decomposition . In this study, in the preparation of liposomes by adding fluorescein, pre-experiment found that the
existence of drugs and the same luciferase encapsulation probability, the measured fluorescence encapsulation
efficiency and drug encapsulation efficiency are basically the same, so the present study, fluorescence spectroscopic Determination of drug entrapment efficiency.
Previous paper  have reported on the HB-? a freeze-
dried liposome size and distribution of research, this section mainly elaborated on the HB in different freeze-drying
protective agents effective in the encapsulation efficiency of liposome composition, appearance shape, grain Drive the impact of the establishment of a stable lipid formulations and liposome storage, packaging, transportation, and so opened up a good way.
An instrument and reagent
1.1 Instrument SENCO R series rotary evaporator (Shanghai Nobuo Technology Co., Ltd.), JY92-? ultrasonic cell
pulverizer (Bio-Technology Co., Ltd. Ningbo Xin-zhi), Z233MK-2
low-temperature high-speed centrifuge (HERMLE), F-3000
fluorescence spectrophotometer (HITACHI), AR2140 electronic analytical balance (Ohaus Corporation), SZ-93 Automatic double
pure water distillation device (on the Yahya-wing biochemical
Instrument Factory), DM LB2 microscope (LEICA), QWIN V3 Digital Image Analysis software (LEICA), YRD1501A vacuum freeze-drying machine (installed power: 6.7 kW, Shanghai Yucheng Drying Equipment Co., Ltd.).
1.2 Test drug cholesterol (cholesterol, CH, AR, Shanghai Chemical Reagent Emerging Research Institute, lot number 030,318), Soy Lecithin (Soybean Lecithin, Serva imported packaging and biochemical reagents, Shanghai Boao Bio-
Technology, Inc., Lot 050,314 ), fluorescein (Fluorescein, AR, Shanghai East China Normal University, China factory, Shanghai Jin Ling Fine Chemicals Co., Ltd., batch number 20041212), HB (homemade), sucrose (Sucrose, AR, Shanghai Reagent Factory, batch number 68 -01-06), glycine (Glycine, biochemical
reagent, Shanghai Nanxiang Reagent Co., Ltd., batch number 031.01 thousand), mannitol (Mannitol, AR, Sinopharm Chemical Reagent Co., Ltd., batch number F20050527), sorbitol (D-
Sorbitol, biochemical reagent, Shanghai Reagent Factory),
trehalose (Trehalose, biochemical reagent, Shanghai lava Fine Chemical Co., Ltd., batch number 040,801), polyvinyl pyrrolidone (K30) (Polyvinlpyrrolidone, imports of packaging and Sinopharm Group Chemical Reagent Co., Ltd., batch number F20050531).
Disodium hydrogen phosphate (Na2HPO4 * 12H2O, AR, Shanghai Ling Feng Chemical Reagent Co., Ltd.), sodium dihydrogen phosphate (NaH2PO4 * 2H2O, AR, Jiangsu Taicang Chemical Plant), phosphate buffered saline (PBS) : 15 mmol * L-1 sodium chloride, 0.2 mmol * L-1Na2HPO4 * 12H2O ~ 0.2 mmol
* L-1NaH2PO4 * 2H2O (81:19), pH 7.4.
2 Methods and Results
2.1 freeze-drying protective agent options
Experimental formulation design PC: CH: HB = 500 mg: 250 mg: 100 mg1 Suc: PC: CH: HB = 4:1:0.5:0.22 Suc: PC: CH: HB = 2:1:0.5:0.23 Gly: PC : CH: HB = 4:1:0.5:0.24 Gly: PC: CH: HB = 2:1:0.5:0.25 Man: PC: CH: HB = 4:1:0.5:0.26 Man: PC: CH: HB = 2:1:0.5:0.27 D-Sor: PC: CH: HB = 4:1:0.5:0.28 D-Sor: PC: CH:
HB = 2:1:0.5:0.29 Tre: PC: CH: HB = 4 : 1:0.5:0.210 Tre: PC:
CH: HB = 2:1:0.5:0.211 Suc: Gly: PC: CH: HB =
4:0.8:1:0.5:0.212 Suc: Man: PC: CH: HB = 4:0.8:1:0.5:0.213 Suc: D-Sor: PC: CH: HB = 4:0.8:1:0.5:0.214 Suc: Tre: PC: CH: HB = 4:0.8:1:0.5:0.215 Suc : PVP: PC: CH: HB =
4:0.8:1:0.5:0.216 PC: CH: HB = 1:0.5:0.2
2.2 HB and effective components of liposomes prepared using reverse-phase evaporation, take prescribed amount of PC and CH dissolved in ether, 10 ml; an alternative HB, freeze-
dried protective agent, fluorescein 1ml (8 μg / ml) dissolved
in 5 ml normal saline solution, mixing the two eggplant-shaped
bottle, ultrasonic probe 30 s (each ultrasound 15 s, interval 4 s); 40 ? under vacuum rotary evaporator to remove ether, to become viscous solution and then Add 5 ml normal saline, probe ultrasound 60 s (each ultrasound 15s, intermittent 4s); to
continue to pressure rotary evaporation of 1 h, were active components of liposomes HB Suspension emulsion.
2.3 Freeze-drying process of getting a prescription liposome suspension 1 ml, all three copies, respectively,
placed in 2.5 ml Xilin bottle, the thermocouple located in bottom of the bottle. Pre-freezing process, 2 h, the sample
temperature of -50 ?. Vacuum to maintain vacuum at 10 Pa
around for the first time a drying process (sublimation phase), continuous 17 h, this time the sample temperature from -50 ? to rise to 2.4 ?, shelf temperature maintained at
10 ?. Increasing shelf temperature to 40 ?, for the first
two sub-drying process (analysis phase), continuous 8 h, freeze-drying process is complete. Freeze-drying curve shown
in Figure 1.
Figure 1 freeze-drying curve (omitted)
2.4 Fluorescence Determination of entrapment efficiency
2.4.1 standard curve of fluorescence intensity of the drawing to take appropriate preparation of fluorescein
concentration of 0.8 μg * ml-1 of the mother liquor,
precision drawing 1,10,20,100,1 000 ml mother liquor were placed in 1 000 ml flask, the with re-distilled water diluted
to scale, prepared into a concentration of 0.000 8,0.008,0.016,0.08, 0.8 μg * ml-1 of the series solution with
water as a blank, at Ex = 490 nm; Em = 510 nm wavelength was measured F values to F values and the concentration as the standard curve. The results showed that fluorescein standard solution at 0.000 8 ~ 0.8 μg * ml-1 concentration range of
good linearity, the regression equation: Y = 8 822.8X 310.95, r = 0.999 8. Reposted elsewhere in the paper for free download http://
2.4.2 Preparation of the tested products to take the various prescriptions are not freeze-dried liposome suspension
1 ml, all three were, at T = 10 ?; t = 15 min; V = 15 000 r /
min centrifugation, taking on the clear liquid fixed volume to 5 ml, as for the test product A. After the freeze-dried
samples were re-dissolved PBS, at T = 10 ?; t = 20 min; V =
18 000 r / min centrifugation, the supernatant constant volume to 5ml, as for the test product B.
2.4.3 fluorescence obtained for the test materials A, B solution, with water as a blank, at Ex = 490 nm; Em = 510 nm wavelength was measured F values, and calculate the
encapsulation efficiency. The results shown in Table 1 and Figure 2.
Liposome encapsulation efficiency (%) = (W total-W Tour)
/ W total × 100%
W Total: drug inputs, W travel: the amount of free drug
Table 1 Determination of encapsulation efficiency results (omitted)
Figure 2 variation of freeze-dried before and after the
encapsulation efficiency (abbreviated)
According to a comparison of freeze-dried before and
after the encapsulation efficiency can be drawn: In the sample of freeze-dried encapsulation efficiency improvement and protection of areas: the concentration of protective agents at high concentrations of low protective effect than that good. One good effect of glycine and mannitol, dual protective agents in order to sucrose - glycine, sucrose - two of the
protective effect of trehalose better. Sorbitol and sucrose dual protection agents - mannitol, sucrose - sorbitol somewhat
less protective effect. Substituting sucrose, trehalose and sucrose-PVP least effective protective agent.
2.5 freeze-dried before and after the change in shape and size of freeze-dried after the appearance of: 3,4,5,6 number is white, the rest of Juncheng yellow. To take 16 prescription
of freeze-dried liposomes with PBS, after re-melting, with a
microscope and digital image analysis software for image processing to measure particle size. The results shown in Table 2 and Figure 3.
Table 2 Determination of particle size results (omitted)
Figure 3 Freeze-dried before and after the particle size variation (abbreviated)
According to freeze-dried samples obtained before and
after comparison of particle size: most of the samples were freeze-dried after the average particle size increases, the sample size of the freeze-drying protection, freeze-dried
protective agent as possible, freeze-dried before and after
the change in particle size should also be smaller. From the analysis of the map were: sucrose, mannitol, and sucrose dual
protective agent - glycine, sucrose - a better protective
effect of sorbitol, glycine, trehalose, sucrose - trehalose,
sucrose-PVP protective effect of the second, while the sorbitol and sugar - less protective effect of mannitol.
Before and after the adoption of freeze-dried liposome
encapsulation efficiency and particle size of the comparison, determine the best protective agent for the freeze-dried
mannitol and binary protective agent sugar - glycine, the
encapsulation rate of 69.91 percent and 66.70 percent ratio, respectively, Mannitol: PC: CH: HB = 4:1:0.5:0.2; sucrose: glycine: PC: CH: HB = 4:0.8:1:0.5:0.2.
Early freeze-drying technology is mainly used for the preservation of biological materials, its principle is the use of frozen solution at low temperature under low pressure conditions, from the frozen state directly without passing through the liquid distillation to remove water to complete drying. Freeze-drying technology allows the drug maintain
their physical and chemical properties and physiological activity, and the minimum loss of active ingredients. In addition, the freeze-dried formulations unique porous
structure, can make drugs easily re-re-water recovery
activity, and the freeze-dried formulations with low water
content, easy to save long-term stability. Freeze-dried
preparation of low-eutectic state within a porous structure, quickly dissolves in the oral cavity to release drugs, so the technology is now also used for instant oral preparations.
PVP is a polymer of freeze-dried protective agent, in the study of freeze-dried blood cells frequently used. We have tried PVP as protecting agent used alone, pre-experiment, when
adding PVP after, PVP poor solubility, the solution become
very viscous, probably due to adhesion of some of the PVP fluorescence, making the measured fluorescence intensity of value is too small a lot, thus the calculated rate of the actual level of entrapment, so not using PVP as protecting
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