CN104546729A - Preparation and application of PGJ2 nanoparticles - Google Patents
Preparation and application of PGJ2 nanoparticles Download PDFInfo
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- CN104546729A CN104546729A CN201510033366.3A CN201510033366A CN104546729A CN 104546729 A CN104546729 A CN 104546729A CN 201510033366 A CN201510033366 A CN 201510033366A CN 104546729 A CN104546729 A CN 104546729A
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Abstract
The invention provides a preparation method of PGJ2 nanoparticles. The preparation method provided by the invention comprises the following steps: dissolving drugs and PLGA (Poly(Lactic-co-Glycolic Acid) in water-insoluble acetone, adding the mixture into an aqueous phase containing a stabilizing agent to homogenize, and evaporating an organic phase at low pressure so as to obtain aqueous-phase particles of the drugs. The PGJ2 nanoparticles prepared by the invention can be applied to absorbing a nano-polymer drug-loading carrier PLGA and preparing a lipid signalling molecule 15d-PGJ2 and nanoparticles of delta12-PGJ2; therefore, the disadvantage of being easy to generate drug resistance due to traditional local application of antibiotics is avoided; and simultaneously, because 15d-PGJ2 and delta12-PGJ2 respectively have the disadvantages of resisting inflammation and promoting osteogenesis, influence of local reference of the PGJ2 nanoparticles to tissue inflammation and repairing in an artificial bone defective rat model repairing process is discussed.
Description
Technical field
The invention belongs to oral cavity material preparation field, relate to a kind of material preparation promoting defective bone regeneration and restoration and suppress defective region inflammation to occur, particularly relate to a kind of PGJ
2the preparation of nanoparticle, and application.
Background technology
Periodontal disease is common chronic infective and tissue destructive disease.Due to the sustainable existence of inflammatory environment, periodontal connective tissue destroys and forms periodontal pocket, and periodontal bone destruction causes frontal resorption, loss of tooth, has a strong impact on the quality of life of patient.The final goal of periodontal treatment is exactly rebuild the supporting tissue lost because of periodontal disease, realizes the regeneration of periodontal.Periodontal regenerative is a complicated biological phenomena, realize periodontal tissue and again recover gum tooth to hat side and combine, not only will form new connective tissue attachment, but also will the alveolar bone of tissue regeneration promoting again.The traditional mechanical Sex therapy of periodontal disease is mainly to eliminate the cause of disease, infection control, and the effect for tissue regeneration is very micro-.The therapy of the induction such as bone collection, guided tissue regeneration, somatomedin, topical application ENAMELIN EMD paradenlal tissue regeneration, can make periodontal membrane, cementum and alveolar bone acquisition regeneration to a certain degree, but the limited regeneration capability of alveolar bone.The tissue engineering technique occurred in recent years has certain advantage in periodontal regenerative treatment, but still there is many insoluble difficulties, one of them is exactly the existence of the special inflammatory environment of periodontitis, make the bone graft implant site moment be subject to the invasion and attack of pathogenic microorganism, implantable bone faces the danger absorbed by inflammatory.Therefore, thoroughly to solve alveolar osteitis in periodontal disease and absorb and repair and regeneration issues, periodontal disease must be controlled and promote that alveolar repair is gone forward side by side.
Recent domestic research finds that prostaglandins lipid molecular has potential anti-inflammatory effect.That current research is more is 15d-prostanoid J
2(15-deoxy-
△ 12,14-prostaglandinJ
2, 15d-PGJ
2) and Delta12-prostaglandin J
2(△
12-prostaglandinJ
2, △
12-PGJ
2).Prostaglandin J
2(ProstaglandinJ
2, PGJ
2) be the product of arachidonic acid metabolic, be converted into 15d-PGJ through 2 spontaneous dehydrations
2; When serum albumin exists, PGJ
2△ can be converted into
12-PGJ
2.15d-PGJ
2peroxisome proliferator-activated receptors-γ (Peroxisome proliferator-activatedreceptors, PPAR-γ) endogenic ligand, to rely on by PPAR-γ or PPAR-γ independent mode suppresses the transcription activating of multiple Pro-inflammatory Cytokine gene.Existing a large amount of vivo and vitro experiment so far confirms 15d-PGJ
2there is potential anti-inflammatory property.In addition, 15d-PGJ
2also may participate in the atomization of osteoblast and osteoclast.△
12-PGJ
2with 15d-PGJ
2structure very similar, but be formation and the metabolism of bone unlike the former Main Function.Research finds, △
12-PGJ
2hydroxyapatite deposition, stimulation alkaline phosphatase activities can be induced in osteoblasts cultivation system, promote the formation of collage synthesis and increase bone tuberosity; △
12-PGJ
2there is the effect of raising resorption lacunae and BGP level in preosteoblast MC3T3-E1 cell; Topical application △ is found in rat animal experiment
12-PGJ
2can dose-dependent mode promote in artificial bone defective region and the formation of the new bone of bone regeneration around implant, and with some somatomedin, as, BMP-2/6 (Bone morphogenetic protein-2/6, BMP-2/6), the expression of platelet-derived growth factor-A/B (platelet-derived growthfactor-A/B, PDGF-A/B) is increased.But, 15d-PGJ
2and △
12-PGJ
2belong to lipid molecular, biological property is unstable, the half-life is short, affects its extensive use in clinical.
Summary of the invention
One of the object of the invention is to provide a kind of PGJ
2the preparation method of nanoparticle---emulsification mechanism, by medicine and the water-soluble immiscible acetone of PLGA, is joined in the aqueous phase being added with stabilizing agent and is homogenized, under lower pressure, evaporated organic facies, obtained an aqueous phase prose style free from parallelism for medicine.Realized by following steps:
(1) poly lactic-co-glycolic acid (Poly (lactic-co-glycolic acid), PLGA) (50:50, m.w.50000g/mol) is taken, 100mg; Class 60,40mg of department; Tristerin, 200mg; Fully dissolve in 30mL acetone, form oil phase, get 1mL for subsequent use;
(2) Tween 80 is taken, 60mg; Fully be dissolved in 30mL distilled water, form aqueous phase, get 1mL for subsequent use;
(3) Delta12-prostaglandin J is added in 1mL oil phase mixed liquor
2(△
12-prostaglandinJ
2, △
12-PGJ
2), mix after 100 μ g, slowly add in 1mL aqueous phase, limit edged stirs, Keep agitation 10 points;
(4) mixed liquor that finally obtains of rotary evaporation, evaporation acetone wherein, final 15d-PGJ
2/ △
12-PGJ
2drug level be 100mg/L.Final liquid volume 1mL, wherein 15d-PGJ
2/ △
12-PGJ
2total amount be 100ug, therefore final 15d-PGJ
2/ △
12-PGJ
2drug level be 100mg/L.
Blank nanoparticle presses same method preparation.
The nanoparticle prepared carries out granularmetric analysis, transmission electron microscope observing, carrying drug ratio and extracorporeal releasing test and comprehensively evaluates its physicochemical property.
Another object of the present invention is to provide the PGJ of above-mentioned preparation
2nanoparticle is as the application in pharmaceutical carrier.15d-PGJ
2and △
12-PGJ
2belong to lipid molecular, except the shortcomings such as biological property is unstable, the half-life is short, the water solublity of the two is extremely low, is applied to after in animal body and only has few part can enter target cell performance effect.Its slow-release function can prolong drug circulation time in vivo, improves the pharmacokinetics of medicine.On the one hand, be that carrier extends action time in half-life of medicine and body with PLGA; On the other hand, PLGA is as 15d-PGJ
2and △
12-PGJ
2carrier, the uptake ratio of medicine can be improved, improve the functioning efficiency of medicine.
The main process related in periodontal tissue's inflammatory reaction comprises: host immune inflammatory reaction, and matrix metalloproteinase produces, arachidonic acid metabolite, frontal resorption.Conventional medicine mainly contains: IL-1 and TNF receptor antagonist, can suppress absorption and the periodontal attachment loss of alveolar bone; Low dose of tetracycline whole body application can suppress the activity of matrix metalloproteinase; The whole body application of NSAID (non-steroidal anti-inflammatory drug) can pass through number of ways, reduces metabolism and the synthesis of prostaglandin, reduces bone resorption.Compared with said medicine, 15d-PGJ
2for regulating the inflammatory reaction of patients with periodontal disease, there is certain advantage as the endogenous lipid molecule in body: 15d-PGJ
2possesses the physiological function of above-mentioned several drugs simultaneously; Compared with the antiinflammatory action of COX-2 enzyme inhibitor class medicine (as: aspirin), 15d-PGJ
2the activity of COX-2 can not be suppressed completely, remain the antiinflammation of COX-2 enzyme in the inflammation later stage.
Research finds, △
12-PGJ
2can induce hydroxyapatite deposition, stimulation alkaline phosphatase activities, promote the formation of collage synthesis and increase bone tuberosity, raise some somatomedin, e.g., the expression of BMP-2/6, PDGF-A/B, promotes in artificial bone defective region and the formation of the new bone of bone regeneration around implant.
PLGA polymer is a kind of degradable functional polymer organic polymer.In vivo, the degraded of PLGA by being break to form monomer acids, lactic acid and hydroxyacetic acid by ester bond, and then is eliminated in vivo.The final hydrolyzate of PLGA is carbon dioxide and water, and lactic acid is also normal sugar metabolism product in body, therefore has good biocompatibility, nontoxic, non-stimulated, the characteristic such as non-immunogenicity and medicament slow release.The above-mentioned characteristic of nano-high molecule carrier makes it extremely be applicable to the carrier of periodontal local application and periodontal tissue various somatomedin in sound, however at present domestic about for periodontal local Nano medication research be still blank.The minocycline control release type local application that many employings are carrier with Absorbable rod Nano microsphere on U.S.'s periodontal disease clinical treatment, commodity are called " Arestin ", but said medicine is broad ectrum antibiotic, and life-time service easily causes drug resistance and dysbacteriosis etc.
The present invention's application Absorbable rod nano-high molecule medicine carrying carrier PLGA carrier, builds lipid signal molecule 1 5d-PGJ
2and △
12-PGJ
2nanoparticle, on the one hand avoid traditional topical application antibiotic easily to produce the shortcoming of drug resistance, simultaneously in conjunction with 15d-PGJ
2and △
12-PGJ
2both have the shortcoming of antiinflammatory and short ossification respectively, inquire into the impact of tissue inflammation and reparation in the rat model repair process quoting artificial bone's defect in its local.
Accompanying drawing explanation
Fig. 1-1 is △ under transmission electron microscope
12plain J is thanked in-prostatitis
2the form of nanoparticle.Present spherical, be uniformly distributed.
Fig. 1-2 is △
12-prostaglandin J
2the Cumulative release amount of nano suspending liquid in 6 hours.0.5,1,2,4,6 constantly little, △
12-prostaglandin J
2the average cumulative release medication amount of nanoparticle is respectively 30 ℅, 52 ℅, 77 ℅, 91 ℅, 98 ℅.
Fig. 1-3 is normal saline (S), blank nanoparticle (K), △
12-prostaglandin J
2(F), △
12-prostaglandin J
2nanoparticle (N) acts on rat bone defective region, when postoperative 3 (A), 7 (B), 14 (C), 28 (D) sky, the Western Blot result of BMP-6 (BMP-6) and EphrinB2 in defective region, GAPDH is internal reference.
Fig. 1-4 be normal saline (a, b, c, d), blank nanometer (e, f, g, h), △
12-prostaglandin J
2(△
12-PGJ
2) (i, j, k, l), △
12-prostaglandin J
2nanoparticle (△
12-PGJ
2-NC) (m, n, o, p) act on the new bone rege-neration in Cranial defect district respectively.Within postoperative 3 days, (be full of blood clot and loose connective tissue in a, e, i, m) defective region, within postoperative 7 days, (b, f, j, n) connective tissue are more and finer and close, △
12-PGJ
2, △
12-PGJ
2the nearly pulp cavity side ,-NC zone of action has immature bone tuberosity to be formed, and within postoperative 14 days, (c, g, k, o) bone tuberosity quantity increase, and within postoperative 28 days, (d, h, l, p) bone structure are ripe.HE dyes, 100 ×, be Cranial defect region shown between two arrows.
Fig. 2-1 is 15d-prostaglandin J under transmission electron microscope
2the form of nanoparticle.Be uniformly distributed, the particle size values comparatively recorded is smaller.
Fig. 2-2 is 15d-prostaglandin J
2the Cumulative release amount of nano suspending liquid in 360 minutes.
When Fig. 2-3 is postoperative 1 (A), 3 (B), 7 (C), 14 (D) sky, normal saline (S), blank nanoparticle (K), 15d-prostaglandin J
2(F), 15d-prostaglandin J
2nanoparticle (N) acts on Cranial defect district, the Western Blot result of interleukin-6 (IL-6), interleukin-11-beta (IL-1 β) and TNFalpha (TNF-α) in surrounding soft tissue, GAPDH is internal reference.
Fig. 2-4 be normal saline (a, b, c, d), blank nanoparticle (e, f, g, h), 15d-prostaglandin J
2(i, j, k, l) and 15d-prostaglandin J
2nanoparticle (m, n, o, p) act on Cranial defect district respectively, postoperative 3 (a, e, i, m), 7 (b, f, j, n), 14 (c, g, k, o) He 28 (the new bone formation situations in d, h, l, p) sky Cranial defect district.Arrow line is the border for defective region.Masson ' s Trichrome dyes.100×。
Fig. 2-5 is the immunohistochemical stainings of EphrinB2 in defective region.Postoperative, normal saline group (S) (a, b, c, d), blank nanometer group (e, f, g, h), 15d-prostaglandin J
2(i, j, k, l) 15d-prostaglandin J
2nanoparticle (m, n, o, p) group after surgery 3 (a, e, i, m), 7 (b, f, j, n), 14 (c, g, k, o) He 28 (the protein expression situations of EphrinB2 in d, h, l, p) Cranial defect district.400×。
Fig. 2-6 is immunohistochemical stainings of osteoprotegerin in defective region (OPG).Postoperative, normal saline group (S) (a, b, c, d), blank nanometer group (e, f, g, h), 15d-prostaglandin J
2(i, j, k, l) 15d-prostaglandin J
2nanoparticle (m, n, o, p) group after surgery 3 (a, e, i, m), 7 (b, f, j, n), 14 (c, g, k, o) He 28 (the protein expression situations of OPG in d, h, l, p) Cranial defect district.400×。
Detailed description of the invention
The present invention is further described with accompanying drawing in conjunction with the embodiments.
Embodiment 1:15d-PGJ
2and △
12-PGJ
2the preparation method of PLGA nanoparticle:
(1) take PLGA compound, 100mg, class 60,40mg of department, tristerin, 200mg, is fully dissolved in 30mL acetone, forms oil phase, gets 1mL for subsequent use;
(2) take Tween 80,60mg, fully dissolve in 30mL distilled water, form aqueous phase, get 1mL for subsequent use;
(3) 15d-PGJ is added in 1mL oil phase mixed liquor
2/ △
12-PGJ
2, 100 μ g, mixing, slowly adds in 1mL aqueous phase, and limit edged stirs, Keep agitation 10 points;
(4) mixed liquor that finally obtains of rotary evaporation, evaporation acetone wherein, final 15d-PGJ
2/ △
12-PGJ
2drug level be 100mg/L.
Embodiment 2.15d-PGJ
2and △
12-PGJ
2the mensuration of physicochemical property of PLGA nanoparticle:
1. assay method:
(1) dynamic light scattering principle measures nanoparticle particle diameter: the nano suspending liquid prepared is diluted 100 times by volume, and nanometer particle size analyser is measured, and result gets 3 meansigma methodss measured.
(2) form of transmission electron microscope observing nanoparticle: by volume suspension is diluted 100 times, copper sheet dips a little and sucks surplus liquid with filter paper, and 1 ℅ phosphotungstic acid dyeing, after natural air drying, observes under transmission electron microscope.
(3) the centrifugal and high efficiency liquid phase chromatographic analysis method (High performance liquid chromatography analysis, HPLC) of ultrafiltration measures carrying drug ratio and the release in vitro behavior of nanoparticle:
1) high performance liquid chromatography is first used to draw variable concentrations: 15d-PGJ
2and △
12-PGJ
2the standard curve of standard substance in methanol solution: during HPLC working sample, select the chromatographic column of C18 (anti-phase, 5 μm, 110A, 150 × 4.60mm), 25 DEG C, mobile phase is made up of 17mM phosphoric acid/acetonitrile (v/v=50/50), filters before using through 0.22 μm of oil film, each injection 20 μ L samples, flow velocity is 1mL/min, is to record peak area under the condition of 230nm at wavelength.According to concentration and the peak area recorded of standard substance, draw 15d-PGJ
2/ △
12-PGJ
2standard curve in methanol solution,
2) carrying drug ratio measures: by nano suspending liquid high speed centrifugation, get supernatant, measure wherein 15d-PGJ
2/ △
12-PGJ
2amount M
1, according to formula, envelop rate=(M
always-M
1)/M
always℅, calculates carrying drug ratio.Repeat 3 times, average.
3) mensuration of release in vitro behavior: get 1mL △ with the bag filter that relative molecular mass is 1000
12-PGJ
2-NC suspension is placed in the centrifuge tube of 15mL, add 5mL PBS (pH=7.2) shaken well and put into constant-temperature shaking incubator (37 DEG C after sealing, 70r/min), add the same PBS of 5mL after collecting the every sub-sampling of sample 5mL. at 30,60,120,240,360 points respectively, continue above-mentioned steps.Experiment repetition 3 times.15d-PGJ in HPLC working sample
2/ △
12-PGJ
2concentration.According to the 15d-PGJ drawn
2/ △
12-PGJ
2concentration and the equation of peak area, 15d-PGJ in calculation sample
2/ △
12-PGJ
2concentration.Take time as abscissa, Cumulative release amount is vertical coordinate, and the burst size that in drawing 360 points, each time point adds up accounts for the release profiles of total burst size.
2. measurement result:
(1) △
12-PGJ
2the physicochemical property of-NC:
△
12-PGJ
2the opalescence white suspension of-NC in homogenizing.Record mean diameter for (135.2 ± 0.85) nm; Transmission Microscopic observation, △
12-PGJ
2-NC is rounded, is uniformly distributed, without a large amount of clustering phenomena (Fig. 1-1); Carrying drug ratio is 92.03%; In extracorporeal releasing experiment, △
12-PGJ
2-NC is Cumulative release amount (Fig. 1-2) in 360 points.
(2) 15d-PGJ
2the physicochemical property of-NC:
15d-PGJ
2– NC is shown as the opalescence white suspension of homogenizing.Record mean diameter for (115.2 ± 0.65) nm; Images of transmissive electron microscope is shown in Fig. 2-1, spherical, and the particle diameter that naked eyes sight particle diameter comparatively records is slightly little; Carrying drug ratio 64%; Fig. 2-2 is shown as 15d-PGJ
2the average cumulative burst size of-NC each time point in 360 minutes.
The foundation of embodiment 3. rat bone defect model
Select cleaning grade Wistar male rat, simple randomization grouping is done to all experiments rat, namely first rat is numbered, then randomly draws, each 12, be divided into 4 groups, be defined as S group, K group, N group and F group respectively.Often organize rat to sort from small to large by number, every 3 is a subgroup, and above-mentioned often group rat was divided into D3, D7, D14 and D28 group according to postoperative 3,7,14,28 days.22 DEG C, 40 ﹪ humidity, 12h daylight is in proper order under condition, and free diet, drinking-water, after fed standard chow 3-7 days, carry out the preparation of Cranial defect model.Pentobarbital sodium (50mg/kg) intraperitoneal injection of anesthesia.After anesthesia, reject the hair at femur position, the rat hindlimb back side, 75 ﹪ alcohol disinfectings, are about the skin incision of 3cm along femur major axis, and subsequently, blunt separation Musclar layer, peels off periosteum, exposes surface of bone.Slowly be drilled in femur stage casing by dentistry and do 5 × 1.5mm Cortical bone deficiency along major axis, constantly with normal saline flushing pre-cooled in a large number in process.After having prepared, make carrier with the absorbability collagen sponge of 5mm × 1.5mm size, infiltrate 30 μ L normal saline (S group), blank nanoparticle (K group), △ respectively
12-PGJ
2-NC/15d-PGJ
2-NC (N group) and △
12-PGJ
2/ 15d-PGJ
2(F group), is placed on Cranial defect district, fixes by the arc titanium sheet of previously prepared long 8mm, and muscle, skin layering are sewed up.
The zoopery of embodiment 4. pharmacology
1. △
12-PGJ
2the coherent detection of nanoparticle and result:
Postoperative 3,7,14,28 days, heavy dose of pentobarbital sodium lumbar injection, row 4 ﹪ paraformaldehyde heart perfusion.Each time point four groups of rats respectively get 3, complete separation femur, then rapid left side specimen is transferred in liquid nitrogen,-80 DEG C of preservations, detect for real-time quantitative-polymerase chain reaction (Real time quantitive-polymerase chain reaction, qRT-PCR), western blotting.Right side is placed in 4 ﹪ paraformaldehyde internal fixtion, for making pathological section, carries out HE staining analysis.
(1) mrna expression of Cranial defect district BMP-6 and PGDF-B
QRT-PCR testing result shows, △
12-PGJ
2and △
12-PGJ
2-NC group, in osseous tissue, the mrna expression of BMP-6 and PGDF-B increases, and each group difference is in table 1.As shown in Table 1, S group is as blank group, and by comparison, different time points, the equal no significant difference of mrna expression of BMP-6 and PGDF-B, result does not have statistical significance (p >=0.05) to K group.By comparison, the mrna expression of BMP-6 and PGDF-B raises F, N group.Wherein, △
12-PGJ
2-NC group raises more remarkable, and result has statistical significance (p < 0.05 or p < 0.001).F, N group is compared with K group, and the mrna expression of BMP-6 and PDGF-B increases.N group compared with F group, △
12-PGJ
2-NC is more obvious to the rise effect of BMP-6 and PGDF-B, has statistical significance (p < 0.05 or p < 0.001).
The relative expression quantity of rat postoperative different time points BMP-6 and PDGF-BmRBA respectively organized by table 1.
Note: S, K, F, N group is normal saline, blank nanoparticle, △ respectively
12-prostaglandin J
2, △
12-prostaglandin J
2nanoparticle acts on rat bone defective region;
a: P < 0.05 compared with S group;
b: P < 0.05 compared with K group;
c: P < 0.05 compared with F group;
a+: P < 0.001 compared with S group;
b+: P < 0.001 compared with K group;
c+: P < 0.001 compared with F group.
(2) protein expression of Cranial defect district BMP-6 and EphrinB2
The protein expression situation of BMP-6 and EphrinB2 is shown in Fig. 1-3.△
12-PGJ
2and △
12-PGJ
2-NC acts on Cranial defect district, increases the expressing quantity of BMP-6 and EphrinB2, △
12-PGJ
2the effect of-NC is more remarkable.But same time point, K group is no significant difference compared with S group.
(3) the new bone formation amount in Cranial defect district
Histological observation, postoperative 3 days, defect area was full of hemocyte and loose connective tissue, does not have bone tuberosity.Postoperative 7 days, loose connective tissue became fine and close connective tissue, has jejune bone tuberosity therebetween.Within postoperative 14 days, there is a small amount of ripe new bone formation defective region, postoperative 28 days, and the newborn bone amount in defective region increases (see Fig. 1-4).The area of new bone component analysis of 14,28 days of ImageJ2X software measurement is in table 2.
The newborn bone amount in rat postoperative 14,28 days Cranial defect districts respectively organized by table 2.
Note: S, K, F, N group is normal saline, blank nanoparticle, △ respectively
12-prostaglandin J
2, △
12-prostaglandin J
2nanoparticle acts on rat bone defective region;
a: P < 0.05 compared with S group;
b: P < 0.05 compared with K group;
c: P < 0.05 compared with F group.
a+: P < 0.001 compared with S group;
b+: P < 0.001 compared with K group.
2.15d-PGJ
2the coherent detection of nanoparticle and result:
Postoperative 1,3,7,14 day, lethal dose pentobarbital sodium lumbar injection.Each time point four groups of rats respectively get 3, and qRT-PCR detects the gene expression amount of IL-6, IL-1 β, TNF-α, BMP-6 and PDGF-B in defective region; Get before being separated femur and close on defective region soft tissue, the Western Blot that homogenate is used for IL-6, IL-1 β and TNF-α protein expression analyzes.
Postoperative 3,7,14,28 days, lethal dose pentobarbital sodium lumbar injection, complete separation femur, is placed in 4 ﹪ paraformaldehyde internal fixtion, makes pathological section, and the new bone formation amount of defective region is observed in row Masson trichroism (Masson trichrome) dyeing; Meanwhile, the protein expression situation of osteoprotegerin (Osteoprotegerin, OPG) and EphrinB2 in row Immunohistochemical detection defective region.
(1) protein expression of IL-6, IL-1 β and TNF-α in Cranial defect district surrounding tissue
Postoperative, the protein expression level respectively organizing IL-6, IL-1 β and TNF-α is shown in Fig. 2-3.As Figure 2-3, postoperative 3 days, the immunity exposure band of N group IL-6, IL-1 β and TNF-α presented low expression; From postoperative 7 days to 28 days, the expression intensity of the above-mentioned factor of N group continued to reduce, and even Western Blot result presents and do not express; During F group 7,14 days after surgery, IL-6, IL-1 β and TNF-alpha expression reduces, and when postoperative 28 days, Western Blot presents and do not express; K group and S group, reduce, but the two compares no significant difference from the expression of postoperative 3 days to the 28 days above-mentioned factors.
(2) mrna expression of IL-6, IL-1 β and TNF-α in Cranial defect district
In Ge Zu Cranial defect district, the mrna expression situation of IL-6, IL-1 β and TNF-α is as table 3.The mrna expression level of F group and N group remarkable IL-6, IL-1 β and TNF-α significantly reduces, and the downward effect of the latter is more obvious, and result difference has statistical significance (p < 0.05).Prompting 15d-PGJ
2and 15d-PGJ
2-NC can lower the gene expression of above-mentioned inflammatory factor.
The mRNA relative expression quantity of IL-6, IL-1 β and TNF-α in table 3. postoperative different time points Cranial defect district
Note: S, K, F, N group is normal saline, blank nanoparticle, 15d-prostaglandin J respectively
2, 15d-prostaglandin J
2nanoparticle acts on rat bone defective region;
a: P < 0.05 compared with S group;
b: P < 0.05 compared with K group;
c: P < 0.05 compared with F group.
(3) gene expression of BMP-6 and PDGF-B in Cranial defect district
As shown in table 3, compared with S group, the gene expression amount of N group BMP-6 and PDGF-B significantly increases and has statistical significance (p < 0.05).F group, the expression of BMP-6 and PDGF-B also raises to some extent, but effect is remarkable not as N group, and result has or do not have statistical significance.Postoperative 14 days, the most obviously, the increase of prompting osteogenic activity may be relevant to alleviating of inflammatory reaction for N group BMP-6 and PDGF-B gene upregulation.S with K group is compared, the equal no significant difference of expression of BMP-6 and PDGF-B.
The relative expression quantity of BMP-6 and PDGF-BmRNA in table 4. postoperative different time points Cranial defect district
Note: S, K, F, N group is normal saline, blank nanoparticle, 15d-prostaglandin J respectively
2, 15d-prostaglandin J
2nanoparticle acts on rat bone defective region;
a: P < 0.05 compared with S group;
b: P < 0.05 compared with K group;
c: P < 0.05 compared with F group.
(4) the newborn bone amount in Cranial defect district
As in Figure 2-4, postoperative 3 days, defective region was full of blood clot and loose connective tissue, does not have bone tuberosity to be formed.Postoperative 7 days, in Cranial defect district, connective tissue was finer and close, and a small amount of jejune bone tuberosity appears in nearly pulp cavity side, and after one week, the quantity of bone tuberosity increases.Until 28 days time, Cranial defect region bone structure is more ripe.Postoperative 14, the semi-quantitative analysis of 28 days defective region area of new bone is in table 5.
The newborn bone amount in postoperative 14,28 Tian Shi Cranial defect districts respectively organized by table 5.
Note: S, K, F, N group is normal saline, blank nanoparticle, 15d-prostaglandin J respectively
2, 15d-prostaglandin J
2nanoparticle acts on rat bone defective region;
a: P < 0.05 compared with S group;
b: P < 0.05 compared with K group;
c: P < 0.05 compared with F group.
(5) 15d-PGJ
2-NC raises the protein expression of EphrinB2 and OPG in defective region
As Fig. 2-4, Fig. 2-5 display, the cell of expressing EphrinB2 or OPG presents brown in immunostaining process, is positive staining district.15d-PGJ
2-NC significantly increases the positive stained area in immunostaining, and result has statistical significance (p < 0.05). and the positive staining district of more EphrinB2 and OPG also appears in F group laboratory animal specimen compared with S group.But result not all has statistical significance.During EphrinB2 and OPG 3,7,14,28 days after surgery, the sxemiquantitative statistic analysis result of expression is shown in and table 5.
The semi-quantitative analysis of EphrinB2 and OPG protein expression in table 6. postoperative different time points Cranial defect district
Note: S, K, F, N group is normal saline, blank nanoparticle, 15d-prostaglandin J respectively
2, 15d-prostaglandin J
2nanoparticle acts on rat bone defective region;
a: P < 0.05 compared with S group;
b: P < 0.05 compared with K group;
c: P < 0.05 compared with F group.
Claims (3)
1. a PGJ
2the preparation method of nanoparticle, is characterized in that, is realized by following steps:
(1) take 50:50, m.w.50000g/mol poly lactic-co-glycolic acid, class 60 of department, tristerin, fully dissolve in 30mL acetone, forms oil phase, get 1mL for subsequent use;
(2) get Tween 80, be fully dissolved in distilled water, form aqueous phase, get 1mL for subsequent use;
(3) in 1mL oil-phase solution, add 100 μ g △
12-prostaglandin J
2rear mixing, slowly adds in 1mL aqueous phase, and limit edged stirs, Keep agitation 10 points;
(4) mixed liquor that obtains of rotary evaporation step (3), evaporation acetone wherein, final 15d-prostaglandin J
2/ △
12-prostaglandin J
2drug level be 100mg/L.
2. a kind of PGJ according to claim 1
2the preparation method of nanoparticle, is characterized in that, final liquid volume 1mL, wherein 15d-PGJ
2/ △
12-PGJ
2total amount be 100ug, therefore final 15d-PGJ
2/ △
12-PGJ
2drug level be 100mg/L.
3. a kind of PGJ of preparing of method according to claim 1
2nanoparticle is as the application in pharmaceutical carrier.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2010005740A2 (en) * | 2008-06-16 | 2010-01-14 | Bind Biosciences, Inc. | Methods for the preparation of targeting agent functionalized diblock copolymers for use in fabrication of therapeutic targeted nanoparticles |
| CN102218068A (en) * | 2011-04-01 | 2011-10-19 | 上海市肿瘤研究所 | Application of PPARgamma stimulant 15d-PGJ2 to preparation of targeting liver cancer stem cell drugs |
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2015
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|---|---|---|---|---|
| WO2010005740A2 (en) * | 2008-06-16 | 2010-01-14 | Bind Biosciences, Inc. | Methods for the preparation of targeting agent functionalized diblock copolymers for use in fabrication of therapeutic targeted nanoparticles |
| CN102348468B (en) * | 2009-07-31 | 2014-11-05 | 西安力邦医药科技有限责任公司 | Nanosphere or microsphere drug carrier, preparation method, composition and use thereof |
| CN102218068A (en) * | 2011-04-01 | 2011-10-19 | 上海市肿瘤研究所 | Application of PPARgamma stimulant 15d-PGJ2 to preparation of targeting liver cancer stem cell drugs |
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