CN114931650A - Quantum dot nano-drug carrier modified based on dihydrolipoic acid and preparation method and application thereof - Google Patents

Quantum dot nano-drug carrier modified based on dihydrolipoic acid and preparation method and application thereof Download PDF

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CN114931650A
CN114931650A CN202210572144.9A CN202210572144A CN114931650A CN 114931650 A CN114931650 A CN 114931650A CN 202210572144 A CN202210572144 A CN 202210572144A CN 114931650 A CN114931650 A CN 114931650A
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quantum dot
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drug carrier
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赵美霞
任斌
刘永芳
李林松
赵雪杰
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Henan University
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Abstract

The invention belongs to the field of chemistry and biology, and particularly relates to a quantum dot nano-drug carrier modified based on dihydrolipoic acid, and a preparation method and application thereof. The quantum dot nano-drug carrier modified based on the dihydrolipoic acid has the following general formula:

Description

Quantum dot nano-drug carrier modified based on dihydrolipoic acid and preparation method and application thereof
Technical Field
The invention belongs to the technical field of chemical biology, and particularly relates to a quantum dot nano-drug carrier modified based on dihydrolipoic acid, and a preparation method and application thereof.
Background
Quantum Dots (Quantum Dots) are a novel semiconductor fluorescent nano material, mainly composed of II-VI group elements (such as CdSe and CdTe) or III-V group elements (such as InP), and the diameter of the Quantum Dots is between 1 and 100 nm. Compared with the traditional fluorescent dye, the quantum dot has the advantages of longer fluorescence life, higher fluorescence quantum yield, wider excitation spectrum and narrower emission spectrum, and the quantum dot has larger specific surface area and easier surface modification, so that the quantum dot can be widely applied to the biomedical fields of targeted drug loading, disease diagnosis, biological imaging and the like.
Dihydrolipoic acid (DHLA) is an organic compound obtained by hydrogenating lipoic acid, and is a carboxylic acid with two mercapto groups, and it has two enantiomers, but only its R-type enantiomer has biological activity. The DL-lipoic acid used in the experiment is alpha-lipoic acid. DL-lipoic acid is a unique anti-radical substance, commonly referred to as a broad antioxidant. As the mercapto group has strong coordination capacity with metal elements such as Cd and Zn on the surface of the quantum dot, the mercapto group is generally used as a coordination agent for displacement, but the quantum dot coated by the single mercapto group ligand has poor stability. By using the dihydrolipoic acid containing the dimercapto, the stability of the quantum dot in the aqueous solution can be improved.
The polyethylene glycol (PEG) can not only enhance the water solubility and the stability of the nano drug-carrying system, but also avoid the recognition and the non-specific uptake of the nano drug-carrying system by a reticuloendothelial tissue system as far as possible, thereby prolonging the half-life period of the drug-carrying system in the circulation. It has good biocompatibility with many organic matters, can increase certain solubility with compound with poor water solubility, and can inhibit adsorption of transaminase system and reduce its elimination by system when the compound enters blood after being modified by PEG, thereby prolonging blood circulation time and affecting the transport of drug delivery system. Therefore, PEG has been widely studied in the fields of controlled drug release, wound healing, drug delivery, and the like.
The 10-Hydroxycamptothecin (HCPT) is used as anticancer drug carried by the system, and is quinoline alkaloid separated from Camptotheca acuminata of Davidiaceae. It is a specific anti-cancer drug in S phase, and the action mechanism is to inhibit DNA topoisomerase I. Has lower toxicity than camptothecin and no obvious cross drug resistance with common anticancer drugs. However, HCPT cannot be applied effectively clinically due to its poor water solubility and inherent instability caused by opening of an unstable lactone ring under physiological pH conditions, and the existence of the nano-drug carrier perfectly solves this problem.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a quantum dot nano-drug carrier modified based on dihydrolipoic acid and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a quantum dot nano-drug carrier based on dihydrolipoic acid modification has the following general formula:
Figure BDA0003659514910000021
the ligand is dihydrolipoic acid and polyethylene glycol; QDs are CdSe/ZnS quantum dots; n-44-46.
The invention provides a preparation method of the quantum dot nano-drug carrier based on dihydrolipoic acid modification, and the synthetic route is as follows:
Figure BDA0003659514910000022
the method specifically comprises the following steps:
1) dissolving 0.156g of Compound 1 DL-Lipoic Acid (LA) in methanol to give a pale yellow clear transparent liquid, and slowly adding 0.050-0.070g NaBH 4 Reducing and breaking a disulfide bond of the lipoic acid into a dimercapto group, stirring at room temperature for 2-5h, and stopping stirring to obtain a compound 2 DHLA; due to the inverse ofThe yield is high and reaches more than 90 percent, only the product dihydrolipoic acid with double sulfydryl can be modified on the quantum dots, and the raw material medicine DL-lipoic acid and the solvent methanol have no influence on the subsequent reaction and are easy to remove, so that the next reaction can be directly carried out without related separation and purification;
2) adding a proper amount of chloroform solution containing CdSe/ZnS quantum dots into the product obtained in the step 1), stirring for 3-6h in a dark place (the quantum dots can be observed to be transferred to the upper layer, the color of the quantum dots is changed from orange to red, the lower layer is a colorless chloroform layer), transferring the water-soluble quantum dots modified by the upper layer into an EP tube, adding acetone (the water-soluble quantum dots and the acetone can be mixed according to the volume ratio of 1: 2-5), shaking uniformly, centrifuging, discarding the supernatant, collecting the precipitate, and obtaining a pure water-soluble quantum dot 3CdSe/ZnS @ DHLA solid;
3) dissolving 0.100g of the product 3 obtained in step 2) in dimethyl sulfoxide (DMSO), adding 0.350-0.380g of Dicyclohexylcarbodiimide (DCC) and 0.0250-0.040g of 4-Dimethylaminopyridine (DMAP), stirring for 0.5-2h to activate carboxyl, and adding PEG 2000 Stirring at room temperature in dark for 10-24h, transferring the obtained product into an EP tube, adding acetone (the product and the acetone can be mixed according to the volume ratio of 1: 2-5), shaking uniformly, centrifuging, and removing the upper layer liquid to obtain the pure product 4CdSe/ZnS @ DHLA-PEG.
Specifically, the mass ratio of the CdSe/ZnS quantum dots to the compound 2 in the step 2) is 1: 6-8, wherein the volume ratio of the modified quantum dots to acetone is 1: 2-5.
Specifically, the product 3 and PEG in the step 3) 2000 The mass ratio of (A) to (B) is 1: 6-10.
The invention provides a method for loading a drug by using the nano drug carrier, which comprises the following steps:
Figure BDA0003659514910000031
the method specifically comprises the following steps:
a) taking 0.040g of compound 6 succinic anhydride, adding anhydrous Dimethylformamide (DMF), stirring and dissolving, adding 0.030-0.040g of 4-lutidine (DMAP), continuously stirring for 0.5-2h to carry out ring opening and carboxyl activation of anhydride, then adding a compound 510-Hydroxycamptothecin (HCPT), stirring for 24-36h at room temperature, adding a methanol water solution, continuously stirring for 0.5-2h to hydrolyze excessive anhydride, adding methanol into the obtained yellow liquid, standing for 12-24h at 0-8 ℃, after crystallization, collecting a product, vacuum drying and carrying out column chromatography purification to obtain a purified compound 7 cHCPT;
b) dissolving 0.100g of compound 7cHCPT in anhydrous DMSO, adding 0.020-0.030g of Dicyclohexylcarbodiimide (DCC) and 0.035-0.045g of 4-Dimethylaminopyridine (DMAP) to activate carboxyl for 0.5-2h, adding the carrier product 4, stirring at room temperature in the dark for 12-24h, transferring the obtained product into an EP tube, adding acetone (the product and the acetone are mixed according to the volume of 1: 2-5), shaking uniformly, centrifuging, collecting the product, and freeze-drying to obtain a pure product 8(CdSe/ZnS @ DHLA-PEG-HCPT).
Specifically, the molar ratio of the compound 5 to the compound 6 in the step a) is 1: 1-3; the mass ratio of the product 4 to the compound 7 in the step b) is 1: 1-2.
The invention also provides a medicine loaded by the method.
The invention also provides application of the quantum dot nano-drug carrier modified based on the dihydrolipoic acid in drug loading and cell bioactivity.
The invention also provides the application of the medicine in inhibiting the biological activity of cancer cells.
In the above application, the cancer cell may include a cervical cancer cell, a lung cancer cell, a glioma cell, a liver cancer cell, a breast cancer cell, and the like.
The general synthetic route involved in the present invention is as follows:
Figure BDA0003659514910000041
the dihydrolipoic acid modified quantum dots are prepared by reacting dihydrolipoic acid with quantum dots in a chloroform solvent under an alkaline environment, precipitating, centrifugally separating and purifying, and then connecting PEG to ensure that the dihydrolipoic acid modified quantum dots have good biocompatibility and the in-vivo circulation time of the whole system is prolonged. Compared with the prior art, the invention has the following beneficial effects:
1) uv and fluorescence data show: the quantum dots are modified by the dihydrolipoic acid with the dimercapto, so that the modified quantum dots have better water solubility and stability;
2) according to the invention, PEG is connected on the surface of the quantum dot modified by the dihydrolipoic acid, so that the modified quantum dot has good biocompatibility, and the nano material with high biocompatibility is prepared, thereby realizing the effect of the nano material as a nano drug carrier in the field of chemical biology;
3) the preparation method of the quantum dot nano-drug carrier based on the dihydrolipoic acid modification is simple and easy to implement and has lower cost;
4) the quantum dot nano-drug carrier modified by the dihydrolipoic acid has good optical characteristics, can provide good optical signals for carrying and releasing drugs, and provides convenient conditions for tracing the drugs.
Drawings
FIG. 1 is a Transmission Electron Microscope (TEM) map of quantum dots (CdSe/ZnS) and dihydrolipoic acid modified quantum dot carrier 4(CdSe/ZnS @ DHLA-PEG) and drug loaded product 8(CdSe/ZnS @ DHLA-PEG-HCPT) in example 1;
FIG. 2 is the UV absorption spectra of quantum dot carrier 4(CdSe/ZnS @ DHLA-PEG) and product 8(CdSe/ZnS @ DHLA-PEG-HCPT) after modification of quantum dot (CdSe/ZnS) and dihydrolipoic acid in example 1;
FIG. 3 shows the fluorescence spectra of quantum dots (CdSe/ZnS) and dihydrolipoic acid modified quantum dot carrier 4(CdSe/ZnS @ DHLA-PEG) and drug loaded product 8(CdSe/ZnS @ DHLA-PEG-HCPT) in example 1.
Detailed Description
The technical solution of the present invention is further described in detail with reference to the following examples, but the scope of the present invention is not limited thereto.
Name and model of the experimental instrument:
U.S. Perkin-Elmer Lambda-850 UV Spectrophotometer;
U.S. Perkin-Elmer Ls55 spectrofluorometer;
japanese JEOL JEM-200CX transmission electron microscope;
a U.S. BioTek multifunctional microplate reader;
in the following examples, the quantum dots CdSe/ZnS can be prepared by conventional techniques in the art, and the details of the invention are omitted. Reference may be made to the following documents:
F.Pinaud,D.King,H.-P.Moore,S.Weiss.Bioactivation and Cell Targeting of Semiconductor CdSe/ZnS Nanocrystals with Phytochelatin-Related Peptides.J.Am.Chem.Soc.2004,126(19):6115-6123；
X.Peng,J.Wickham,A.P.Alivisatos.Kinetics of II-VI and III-V Colloidal Semiconductor Nanocrystal Growth:“Focusing”of Size Distributions.J.Am.Chem.Soc.1998,120(21):5343-5344。
example 1
1, the synthesis steps for preparing the quantum dot nano-drug carrier modified based on the dihydrolipoic acid are as follows:
Figure BDA0003659514910000051
1) preparation of compound 2: 0.156g of compound 1 (DL-lipoic acid, LA) powder was weighed into a 100mL round-bottom flask, and 20mL of methanol was added thereto and dissolved with stirring to obtain a pale yellow clear transparent liquid. Then accurately weighing 0.060g of NaBH 4 Slowly adding into a bottle, stirring at room temperature for 2h to reduce the disulfide bond of the lipoic acid into bis-sulfhydryl, and stopping stirring to obtain compound 2 (DHLA). Because the reaction yield is higher and reaches more than 90 percent, and only the product dihydrolipoic acid with double sulfydryl can be modified on the quantum dots, the raw material medicine DL-Lipoic Acid (LA) and the solvent methanol have no influence on the subsequent reaction and are easy to remove, the next reaction can be directly carried out without related separation and purification;
2) preparation of compound 3: 20mL of the dihydrolipoic acid liquid synthesized in the last step (methanol solution containing 0.157g of the compound 2) and 2mL of chloroform solution containing 0.020g of CdSe/ZnS quantum dots are mixed and stirred for 3 hours in a dark place, the quantum dots are observed to be transferred to the upper layer, the color of the quantum dots is changed from orange to red, and the lower layer is a colorless chloroform layer. Transferring the quantum dots with the modified upper layer into a 7mL EP tube, mixing the water-soluble quantum dots and acetone according to the volume ratio of 1:2, shaking uniformly, centrifuging for 5min at 10000rpm, removing supernatant, and collecting precipitate to obtain a pure water-soluble quantum dot product 3(CdSe/ZnS @ DHLA) solid;
3) preparing a nano carrier 4: 0.100g of product 3(CdSe/ZnS @ DHLA) was dissolved in 15mL of dimethyl sulfoxide (DMSO), 0.366g of Dicyclohexylcarbodiimide (DCC) and 0.032g of 4-Dimethylaminopyridine (DMAP) were added thereto, and after stirring for half an hour to dissolve them, 0.600g of PEG was added 2000 Stirring at room temperature in dark for 12 h. The resulting product was transferred to a 7mL EP tube, mixed with acetone at a volume ratio of 1:2, shaken well, centrifuged at 12000rpm for 10min, and the supernatant liquid was discarded to give pure product 4(CdSe/ZnS @ DHLA-PEG).
2, the synthesis steps of the dihydrolipoic acid modified quantum dot nano-drug carrier loaded drug are as follows:
Figure BDA0003659514910000061
a) preparation of compound 7: 0.040g (0.0004mol) of Compound 6 (succinic anhydride) was weighed out into a 25mL round-bottomed flask, and 5mL of anhydrous Dimethylformamide (DMF) was added thereto and dissolved with stirring, and 0.035g of 4-lutidine (DMAP) was added thereto and stirring was continued for 0.5h for ring-opening of the acid anhydride and activation of the carboxyl group, after which 0.104g (0.0003mol) of drug 5 (10-hydroxycamptothecin, HCPT) was added and stirred at room temperature for 24 h. Thereafter, 0.5mL of 20% aqueous methanol was added and stirring was continued for 0.5h to hydrolyze excess anhydride. Adding 15mL of methanol into the obtained yellow liquid, standing at 4 ℃ for 12h, collecting the product, vacuum drying at 28 ℃ for 12h, and purifying by column chromatography (V) Methylene dichloride ：V Methanol Gradient elution from 100:1, 50:1, 20:1, 10: 1) to give pure compound 7 (cHCPT);
b) preparation of product 8: after 0.100g of the synthesized compound 7(cHCPT) is dissolved in 15mL of anhydrous DMSO, 0.024g of DCC and 0.041g of DMAP are used for activating carboxyl for 0.5h, and then the carrier product 4 is added according to the mass ratio of the drug compound 7 to the carrier of 1:1, namely 0.100g of CdSe/ZnS @ DHLA-PEG is added, and the mixture is stirred at room temperature for 12h in a dark place. Similarly, the obtained product was transferred to a 7mL EP tube, the product was mixed with acetone at a volume of 1:2, shaken well, centrifuged at 10000rpm for 15min, and the collected product was freeze-dried at-80 ℃ for 48h to obtain pure drug-loaded product 8(CdSe/ZnS @ DHLA-PEG-HCPT).
The sizes and the dispersion degrees of the quantum dot (CdSe/ZnS) and the quantum dot carrier 4(CdSe/ZnS @ DHLA-PEG) modified by the dihydrolipoic acid and the product 8(CdSe/ZnS @ DHLA-PEG-HCPT) after the medicine loading are represented by a transmission electron microscope, and the result is shown in figure 1. As can be seen in fig. 1: the quantum dot carrier 4 modified by the dihydrolipoic acid is uniformly dispersed in water and is spherical, and the particle size of the quantum dot modified by the dihydrolipoic acid is increased from 4.7nm to 5.5nm after the quantum dot is used as a nano-drug carrier to load cHCPT.
The quantum dot carrier 4(CdSe/ZnS @ DHLA-PEG) modified by quantum dots (CdSe/ZnS) and dihydrolipoic acid and the product 8(CdSe/ZnS @ DHLA-PEG-HCPT) after drug loading are characterized by ultraviolet absorption spectrum, and the result is shown in figure 2. As can be seen in fig. 2: the quantum dot carrier 4 modified by the dihydrolipoic acid still has a typical absorption peak of the quantum dot, and the absorption peak of the modified quantum dot is subjected to red shift to different degrees relative to the original quantum dot, which shows that the dihydrolipoic acid has been successfully modified on the quantum dot.
The quantum dot carrier 4(CdSe/ZnS @ DHLA-PEG) modified by quantum dots (CdSe/ZnS) and dihydrolipoic acid and the product 8(CdSe/ZnS @ DHLA-PEG-HCPT) after drug loading are characterized by fluorescence spectrum, and the result is shown in figure 3. As can be seen in fig. 3: after the cHCPT is adsorbed, the emission spectrum shows a slight blue shift, compared with the original quantum dots, the emission spectrum band of the product 8 after drug loading is wider, and the difference of fluorescence between the quantum dots is that the dihydrolipoic acid and the PEG 2000 And the complexation of cHCPT on its surface.
Activity test
Take HeLa: (Human cervical cancer cells), QSG-7701 (human normal liver cells), A549 (human lung cancer cells), SH-SY5Y (human glioma cells), HepG2 (human liver cancer cells), MDA-MB-231 (human breast cancer cells) growth log-phase cells were inoculated into 96-well plates, 90. mu.L/well, 7X 10 cells/well 3 After 24h of cell culture, different concentrations (20-500. mu.g/mL) of the sample dihydrolipoic acid modified quantum dot product 4 (carrier CdSe/ZnS @ DHLA-PEG), compound 7cHCPT and drug loaded product 8(CdSe/ZnS @ DHLA-PEG-HCPT) in example 1 diluted in DMEM medium (containing 10% (v/v) Fetal Bovine Serum (FBS) and 1% (v/v) double antibody (100 units penicillin and 100g streptomycin per mL)) were added in a dosing volume of 10. mu.L and 3 replicates for each drug concentration. Adding drugs for 48h, adding 50 mu LMTT into each hole, acting in an incubator for 4h, then discarding the liquid in each hole, adding 100 mu L dimethyl sulfoxide into each hole to dissolve the bluish purple crystals, and measuring the absorbance at 570nm by using an enzyme-linked immunosorbent assay. Calculating the IC of the sample versus the cells 50 Value (half lethal drug concentration), the cytotoxicity of the samples was evaluated. The results are given in Table 1 below
TABLE 1 cytotoxicity of the vectors prepared in example 1 and the products after loading
Figure BDA0003659514910000081
Table 1 shows that the dihydrolipoic acid modified quantum dot nano-drug carrier 4 and the post-drug-loading product 8 prepared in example 1 have in vitro growth inhibition activity on HeLa, QSG-7701, A549, SH-SY5Y, HepG2 and MDA-MB-231 cells. IC (integrated circuit) 50 The value is the sample concentration value (. mu.g/mL), IC, at which the number of cells capable of normal division growth is suppressed to a level of 50% 50 A larger value indicates a less cytotoxic sample. The experimental results show that: the quantum dot nano-drug carrier 4 modified based on the dihydrolipoic acid has low toxicity to cells and is suitable for serving as a nano-drug carrier; the compound 7cHCPT has an inhibitory effect on cancer cells and normal cells and has a poor effect; the product 8 after drug loading has obviously enhanced inhibition effect on cancer cells, and has lower inhibition effect on normal cells than cancer cells.
To sum up, the following steps are carried out: the dihydrolipoic acid modified quantum dot drug carrier 4 can be used as a nano drug carrier in the field of pharmaceutical chemistry, and can lay a foundation for better designing the nano drug carrier.

Claims (10)

1. A quantum dot nano-drug carrier modified based on dihydrolipoic acid is characterized by having the following general formula:
Figure 458965DEST_PATH_IMAGE001
；
QDs are CdSe/ZnS quantum dots.
2. The preparation method of the quantum dot nano-drug carrier based on the modification of dihydrolipoic acid as claimed in claim 1, wherein the synthetic route is as follows:
Figure DEST_PATH_IMAGE002
the method specifically comprises the following steps:
1) dissolving compound 1 DL-lipoic acid in methanol to obtain light yellow clear transparent liquid, and adding NaBH 4 Stirring at room temperature for 2-5h, and stopping stirring to obtain compound 2 DHLA;
2) adding a chloroform solution containing CdSe/ZnS quantum dots into the product obtained in the step 1), stirring for 3-6h in a dark place, transferring the quantum dots with the modified upper layer into an EP tube, adding acetone, shaking uniformly, centrifuging, removing the supernatant, collecting the precipitate to obtain a water-soluble quantum dot 3CdSe/ZnS @ DHLA solid;
3) dissolving the product 3 obtained in the step 2) in dimethyl sulfoxide, adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine, stirring for 0.5-2h, and adding PEG 2000 Stirring at room temperature in dark for 10-24h, transferring the obtained product to an EP tube, adding acetone, shaking uniformly, centrifuging, and removing the upper layer liquid to obtain the product 4CdSe/ZnS @ DHLA-PEG.
3. The method for preparing a quantum dot nano-drug carrier based on dihydrolipoic acid modification of claim 2, wherein the mass ratio of the CdSe/ZnS quantum dots to the compound 2 in step 2) is 1: 6-8, wherein the volume ratio of the modified quantum dots to acetone is 1: 2-5.
4. The method for preparing a dihydrolipoic acid modification-based quantum dot nano-drug carrier as claimed in claim 2, wherein in the step 3), the product 3 is mixed with PEG 2000 The mass ratio of (A) to (B) is 1: 6-10.
5. A method for loading a drug by using the nano-drug carrier of claim 1, which is characterized in that the synthetic route is as follows:
Figure 563057DEST_PATH_IMAGE003
the method specifically comprises the following steps:
a) adding anhydrous dimethylformamide into compound 6 succinic anhydride, stirring for dissolving, adding 4-dimethylpyridine, continuously stirring for 0.5-2h to perform ring opening of anhydride and carboxyl activation, adding compound 510-hydroxycamptothecin, stirring at room temperature for 24-36h, adding methanol water solution, continuously stirring for 0.5-2h to hydrolyze excessive anhydride, adding methanol into the obtained yellow liquid, standing at 0-8 deg.C, precipitating crystal, collecting product, vacuum drying, and purifying by column chromatography to obtain compound 7cHCPT；
b) Taking out Compound 7cDissolving HCPT with anhydrous DMSO, adding dicyclohexylcarbodiimide and 4-dimethylaminopyridine to activate carboxyl for 0.5-2h, adding carrier product 4, stirring at room temperature in the dark for 12-24h, transferring the obtained product into an EP tube, adding acetone, shaking uniformly, centrifuging, collecting the product, and freeze-drying to obtain the final product.
6. The method for loading a drug by using a nano-drug carrier according to claim 5, wherein the molar ratio of the compound 5 to the compound 6 in the step a) is 1: 1-3; the mass ratio of the product 4 to the compound 7 in the step b) is 1: 1-2.
7. Loading the resulting drug by the method of claim 5 or 6.
8. The application of the dihydrolipoic acid modification-based quantum dot nano-drug carrier of claim 1 in drug loading.
9. The use of the medicament of claim 7 for inhibiting the biological activity of a cancer cell.
10. The use of claim 8, wherein said cancer cells comprise cervical cancer cells, lung cancer cells, glioma cells, liver cancer cells, breast cancer cells.
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Citations (1)

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CN113908286A (en) * 2021-10-14 2022-01-11 河南大学 Polyethyleneimine modified quantum dot nano particle, preparation method thereof and application of quantum dot nano particle as nano drug carrier

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Publication number Priority date Publication date Assignee Title
CN113908286A (en) * 2021-10-14 2022-01-11 河南大学 Polyethyleneimine modified quantum dot nano particle, preparation method thereof and application of quantum dot nano particle as nano drug carrier

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H. TETSUO UYEDA等: "Design of Water-Soluble Quantum Dots with Novel Surface Ligands for Biological Applications", vol. 789, pages 1 - 6, XP008112557 *

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