CN103830738B - A kind of lysine grafting alginate carrier and preparation method thereof - Google Patents
A kind of lysine grafting alginate carrier and preparation method thereof Download PDFInfo
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- CN103830738B CN103830738B CN201410105027.7A CN201410105027A CN103830738B CN 103830738 B CN103830738 B CN 103830738B CN 201410105027 A CN201410105027 A CN 201410105027A CN 103830738 B CN103830738 B CN 103830738B
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Abstract
The present invention relates to a kind of lysine grafting alginate carrier and preparation method thereof.Lysine grafting alginate (ALG-g-Lys) carrier, is characterized in that carrier material is biodegradable, and wherein alginate is polysaccharide, and catabolite can participate in human body repeats itself; Lysine is essential amino acid, has Nutrition and pharmacological effect concurrently.The calcium alginate gel that ALG-g-Lys carrier is relatively simple, stability is stronger, and after genipin is crosslinked, carrier has fluorescent characteristic.The invention provides a kind of novel carrier and substitute alginate as gel rubber material, be applicable to the field such as medicine, food service industry.
Description
Technical field
The present invention relates to a kind of novel lysine grafting alginate (ALG-g-Lys) carrier and preparation method thereof.
Background technology
The Co ntrolled release of medicine also claims controllable drug delivery system, because it has protection and Co ntrolled release speed function to the medicine of embedding or active substance, obtains research and development widely closely during the last ten years.The selection of carrier material is the focus wherein studied, and the carrier material of current use mainly comprises natural, semi-synthetic and synthesis macromolecule three major types, and wherein alginate is a kind of natural hydrogel macromolecular material, because it meets Ca
2+, Ba
2+have the feature of transient gel Deng bivalent cation, the parent being always subject to researcher looks at.
But calcium alginate hydrogel run into chelate compound (phosphate, citrate), non-gelling ion (sodium ion, magnesium ion) or pH value of solution change time, calcium ion in calcium alginate gel is easily replaced or occur swelling, thus makes gel change liquid into gradually and produce the trend of swelling fracture.Therefore, in order to improve the stability of calcium alginate plastic beads, improve the effect of drug controlled release, current researcher mainly takes following two kinds of resolving ideas:
One is utilize its anion characteristic, and wrap one deck cationic polymer thin film or LBL self-assembly formation plural layers at glue pearl skin, this polymeric film has the function of stable calcium alginate plastic beads and regulating drug Co ntrolled release.The filmogen of current employing is a lot, comprises polyamino acid material and chitosan etc.
Another thinking is then directly transform matrix, sodium alginate is carried out modification, introduce new functional group, utilize the interaction of alginic acid macromolecule inter-chain entanglement and cross-linking agent and new functional group to carry out stable aquogel system, as the N-[2-(2-methyl-4-oxopentyl) etc. of sodium alginate.
The present invention utilizes new synthetic material---lysine grafting alginate (ALG-g-Lys) material substitution alginate, and to obtain stability and the good carrier of mechanical strength.
Summary of the invention
In order to obtain a kind of novel carrier, the object of the invention is to new synthetic material lysine grafting alginate (ALG-g-Lys) to be substituted alginate and be used for carrier preparation, this carrier has certain stability and medicament slow release performance.
For realizing object of the present invention, technical scheme of the present invention is: utilize matrix to transform the sodium alginate-modified materials A LG-g-Lys of thinking acquisition, this material maintains the ionic gel performance of sodium alginate, high-pressure electrostatic legal system can be utilized to obtain good sphericity and the glue pearl be evenly distributed.Use cross-linking agent (genipin or glutaraldehyde) cross-linked rubber pearl again, obtain constitutionally stable microsphere.
The preparation technology's flow process that the present invention relates to and concrete operation step as follows:
(1) adopt high-pressure electrostatic method dripping microsphere, ALG-g-Lys solution is instilled CaCl by high voltage electrostatic device by micro-injection pump
2obtained ALG-g-Lys micro gel bead in solution.
(2) from CaCl
2solution leaches micro gel bead, gets 3 times express developed with distilled water, is proceeded in genipin or glutaraldehyde water solution by the micro gel bead collected, and is placed in 37 DEG C of shaking table 60rpm cross-linking reaction 24h.
(3) from solution, microsphere is washed out, with distilled water flushing 3 times.With ethanol gradient elution (60%, 80%, 100%), lyophilization obtains product,
Wherein, the ALG-g-Lys material supply section in step of the present invention (1) is prepared by the following method.
A, in sodium alginate soln, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl), stir, adjustment pH is 5-6; In above-mentioned solution, add N-hydroxy-succinamide (NHS), continue to stir; Add L-lysine hydrochloride (LysHCl); Above-mentioned solution is wrapped up, lucifuge condition lower magnetic force stirring reaction 18-30 hour with masking foil;
B, front solution is transferred in bag filter, is placed in distilled water and dialyses 18-30 hour, be placed in the hydrochloric acid solution of 0.5-1.5mmol/L the 18-30 hour that dialyses, be finally placed in distilled water and dialyse 60-100 hour; The Polyethylene Glycol (PEG20000) that bag filter after having dialysed is placed in high concentration together with solution is interior concentrated; By concentrated solution vacuum lyophilization; Collect dried material, be the ALG-g-Lys material of synthesis.
The ALG-g-Lys carrier glue pearl rounding obtained by the method, smooth surface, particle size distribution is comparatively even, and mean diameter can be controlled between 100 ~ 1000 μm.
In steps A, reactant ratio is n
1: n
2for 1-2:1, n
2: n
3for 1:1, n
1: n
4for 1:1-2, wherein n
1represent the amount of sodium alginate monomeric substance; n
2represent EDCHCl amount of substance; n
3represent NHS amount of substance, n
4represent Lys amount of substance.
In the present invention, step (1) ALG-g-Lys solution concentration (w/v) is preferably for adopting 1.2%-2.0%.
In the present invention, step (1) voltage is preferably for adopting 5.5kv-9.0kv.
The invention has the advantages that:
The feature of lysine grafting alginate (ALG-g-Lys) carrier prepared by the present invention is: carrier material is biodegradable, and wherein alginate is polysaccharide, and catabolite can participate in human body repeats itself; Lysine is essential amino acid, has Nutrition and pharmacological effect concurrently.The calcium alginate gel that ALG-g-Lys carrier is relatively simple, stability is stronger, and after genipin is crosslinked, carrier has fluorescent characteristic.
Detailed description of the invention
First Alg-g-Lys is synthesized
(1) 1.5%(w/v is prepared) sodium alginate 100mL.Take 1.5g sodium alginate with electronic balance, then add 100mL distilled water, be placed on magnetic stirring apparatus and be stirred to sodium alginate dissolving completely.Filter sodium alginate soln with positive press filtration device, the aperture of filter membrane is 0.8 μm and 0.45 μm.
(2) EDCHCl is added.Take EDCHCl1.25g to be dissolved in 15mL distilled water, join in the 100mL sodium alginate soln after filtration, stir, regulate pH to be 5.5.Be placed on magnetic stirring apparatus and stir 30min.
(3) NHS is added.Take NHS0.38g to be dissolved in 10mL water, join above-mentioned solution, be placed in magnetic agitation 30min on magnetic force heating stirrer.
(4) LysHCl is added.Take 1.79gLysHCl(n
lysHCl=1.5n1) be dissolved in 30mL distilled water, join front solution afterwards, with the sodium hydrate regulator solution pH to 7.5 of 1mol/L.
(5) above-mentioned solution is wrapped up with masking foil, lucifuge condition lower magnetic force stirring reaction 24 hours.
(6) be transferred in the good bag filter of advanced processing by front solution, the distilled water being placed in 10L is dialysed 24 hours, is placed in the hydrochloric acid solution of the 1mmol/L of 10L and dialyses 24 hours, and the distilled water being finally placed in 10L is dialysed 72 hours.
(7) concentrated in the PEG20000 bag filter after having dialysed being placed in high concentration (15-20g/L) together with solution.
(8) concentrated solution is placed in ﹣ 20 DEG C of refrigerator pre-freezes, after in ﹣ 80 DEG C of vacuum lyophilizations.Collect dried material, be the ALG-g-Lys material of synthesis.
Embodiment one:
Adopt above operating procedure to prepare carrier, investigate the impact of ALG-g-Lys concentration.Wherein ALG-g-Lys solution concentration be 1.2%, 1.4%, 1.6%, 1.8%, 2.0%(w/v), dissolve with the phosphate buffer of pH7.4.The condition that high-pressure electrostatic prepares glue pearl is: voltage is 7.8kv, and fltting speed is 30mm/h, and syringe needle and liquid level are apart from being 20mm, and syringe needle model is 23G syringe needle, CaCl
2concentration is 2%(w/v).
When ALG-g-Lys concentration is less than 1.6% (comprising 1.6%), glue bead surface is all more smooth; When ALG-g-Lys concentration is greater than 1.6%, glue bead surface is comparatively coarse.When ALG-g-Lys concentration is 1.4%, glue pearl size is comparatively even, and sphericity is better, distortion of almost not trailing.When ALG-g-Lys concentration is 1.4%, glue beadlet footpath is minimum.
Embodiment two:
Concrete operation step is with embodiment one, and difference is the impact investigating voltage, and voltage is set to 5.5kv, 6.0kv, 6.5kv, 7.0kv, 7.5kv, 8.0kv, 8.5kv, 9.0kv.
Be that under the condition of 8.0kv, glue beadlet footpath is minimum at voltage.When voltage is less than 8.0kv, glue beadlet footpath reduces along with the increase of voltage; When voltage is greater than 8.0kv, glue pearl change of size is little.
Embodiment three:
Concrete operation step is with embodiment one, and difference is the impact investigating syringe needle internal diameter, and syringe needle internal diameter is 0.39mm, 0.32mm, 0.27mm, 0.22mm, and corresponding model is respectively 23G, 24G, 25G, 26G.
Syringe needle internal diameter is under 23G condition, and glue pearl is not of uniform size, has conditions of streaking; Syringe needle internal diameter is under 24G condition, and large glue pearl is comparatively even, and small particle is less, trails also less; Syringe needle internal diameter is under 25G condition, and the hangover of glue pearl is more, and in irregular shape more; Syringe needle internal diameter is under 26G condition, and glue pearl is in irregular shape more, and the number of small particle is more.Another syringe needle model is that the glue beadlet footpath of 23G is maximum, and the glue beadlet footpath of 26G is minimum, and general trend is along with the reduction of syringe needle internal diameter, and glue beadlet footpath also reduces.
Embodiment four:
Concrete operation step is with embodiment one, and difference is the impact investigating syringe pump fltting speed, and fltting speed is set to 10mm/h, 20mm/h, 30mm/h, 40mm/h, 50mm/h, 60mm/h.
Syringe pump fltting speed is the very irregular and size very heterogeneity of the glue pearl shape of 10mm/h; Syringe pump fltting speed be the glue pearl size of 20mm/h uneven and have part trail distortion; Syringe pump fltting speed is that the large ball of glue pearl of 30mm/h is comparatively even, and bead is less, trails also less; Syringe pump fltting speed is the glue pearl out-of-shape of 40mm/h, but bead is less; Syringe pump fltting speed is the glue pearl out-of-shape of 50mm/h, size heterogeneity; Syringe pump fltting speed is that the glue pearl hangover of 60mm/h is more, but bead is less.
Embodiment five:
Concrete operation step is with embodiment one, and difference is the impact investigating different solvents, and the pH of phosphate buffer is respectively 5.0,5.8,6.8,7.0,7.4,7.8.
PH5.0 phosphate buffer is that the glue pearl size of solvent is uneven, has few portion deforms; PH5.6 phosphate buffer is that the glue pearl size of solvent is uneven, has most hangover; PH6.8 phosphate buffer is that the glue pearl size of solvent is relatively even, but there is the minimum glue pearl of many particle diameters; PH7.4 phosphate buffer is that the glue pearl size of solvent is even, good sphericity.When solvent is the phosphate buffer of pH7.4, glue beadlet footpath is minimum; When solvent is the phosphate buffer of pH6.8, glue beadlet footpath is maximum; When solvent pH is 5.0 ~ 5.6 time, glue pearl change of size is little; As 6.8<pH<7.4, glue beadlet footpath reduces with the increase of pH value; As pH>7.4, glue beadlet footpath increases with the increase of pH.
Embodiment six:
Concrete operation step is with embodiment one, and difference is to investigate cross-linking agent and concentration impact thereof, and glutaraldehyde concentration (v/v) is 0.1%, 0.4%, 0.8%, 1.2%, and genipin concentration (w/v) is 0.1%, 0.2%, 0.3%, 0.4%, 0.5%.High-pressure electrostatic prepares glue pearl condition: voltage is 8.0kv, syringe needle model 24G, and syringe needle and liquid level distance are 20mm, and fltting speed is 30mm/h, ALG-g-Lys solution concentration is 1.5%(w/v), CaCl
2solution concentration is 1.5% (w/v).
In cross-linking process, microspherulite diameter has obvious expansion.The ALG-g-Lys microspherulite diameter of glutaraldehyde cross-linking is significantly less than the crosslinked ALG-g-Lys microsphere of genipin, and the trend that polymeric microspheres stabilize and crosslinker concentration aggregate performance go out is that polymeric microspheres stabilize increases along with the increase of crosslinker concentration.After genipin is crosslinked, carrier has fluorescent characteristic, and excitation wavelength is 396nm, and emission wavelength is 477nm.
Embodiment seven:
Concrete operation step is with embodiment six, and difference is that methotrexate is dissolved in the PBS buffer of the pH7.4 of ALG-g-Lys.The concentration of methotrexate is respectively 8,4,2mg/mL.
When dispensing concentration is 2mg/ml, drug loading is lower than 2%; When dispensing concentration is 4mg/ml, drug loading is 3% ~ 5%; When dispensing concentration is 8mg/ml, drug loading is between 8% ~ 10%.Envelop rate is between 2% ~ 30%.In 0.5h, the preparation of crosslinked microsphere is all lower than 30%, and release phenomenon without prominent, in 48h, crosslinked microsphere is all greater than 75% to methotrexate preparation, and drug release is substantially complete.
Claims (3)
1. a preparation method for lysine grafting alginate carrier, comprises the steps:
(1) adopt high-pressure electrostatic method dripping microsphere, ALG-g-Lys solution is instilled CaCl by high voltage electrostatic device by micro-injection pump
2obtained ALG-g-Lys micro gel bead in solution;
(2) from CaCl
2solution leaches micro gel bead, gets 3 times express developed with distilled water, is proceeded in genipin or glutaraldehyde water solution by the micro gel bead collected, and is placed in 37 DEG C of shaking table 60rpm cross-linking reaction 24h;
(3) from solution, wash out microsphere, with distilled water flushing 3 times, with ethanol gradient elution, lyophilization obtains product;
Wherein, in step (1), the preparation method of ALG-g-Lys is as follows:
A, in sodium alginate soln, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl), stir, adjustment pH is 5-6; In above-mentioned solution, add N-hydroxy-succinamide (NHS), continue to stir; Add L-lysine hydrochloride (LysHCl); Above-mentioned solution is wrapped up, lucifuge condition lower magnetic force stirring reaction 18-30 hour with masking foil; In steps A, reactant ratio is n
1: n
2for 1-2:1, n
2: n
3for 1:1, n
1: n
4for 1:1-2, wherein n
1represent the amount of sodium alginate monomeric substance; n
2represent EDCHCl amount of substance; n
3represent NHS amount of substance, n
4represent Lys amount of substance;
B, front solution is transferred in bag filter, is placed in distilled water and dialyses 18-30 hour, be placed in the hydrochloric acid solution of 0.5-1.5mmol/L the 18-30 hour that dialyses, be finally placed in distilled water and dialyse 60-100 hour; Concentrated in Polyethylene Glycol bag filter after having dialysed being placed in high concentration together with solution; By concentrated solution vacuum lyophilization; Collect dried material, be the ALG-g-Lys material of synthesis.
2. the preparation method of a kind of lysine grafting alginate carrier as claimed in claim 1, it is characterized in that, step (1) ALG-g-Lys solution concentration w/v is 1.2%-2.0%.
3. the preparation method of a kind of lysine grafting alginate carrier as claimed in claim 1, it is characterized in that, the voltage of step (1) high voltage electrostatic device is 5.5kv-9.0kv.
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| CN108395489A (en) * | 2018-03-22 | 2018-08-14 | 华侨大学 | A kind of 2- nitroimidazoles-cysteine-alginate material and preparation method thereof |
| CN108440684A (en) * | 2018-03-22 | 2018-08-24 | 华侨大学 | A kind of NI-Cys-Alg self-assembled nanometers carrier and its preparation method and application |
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Non-Patent Citations (2)
| Title |
|---|
| "Preparation of microspheres by a novel genipin crosslinking of ALG-g-Lys";Zongxiang Chen et al.;《Journal of Controlled Release》;20131128;第172卷(第1期);第e97页 * |
| "高压微胶囊成型装置制备用于成囊的海藻酸钙胶珠";陈爱政等;《山东生物医学工程》;20021231;第21卷(第04期);第21-23页 * |
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