CN102816331B - Preparation method of polyamidoamine dendritic macromolecular structure modifier and application thereof - Google Patents

Abstract

The invention discloses a preparation method of a polyamidoamine dendritic macromolecular structure modifier and a pharmacy application. The modifier is obtained by covalent coupling of polyamidoamine dendritic macromolecules and a LED209 carboxyl derivative through an amide bond. By the adoption of the polyamidoamine dendritic macromolecular structure modifier, cytotoxicity of PAMAM-NH2 is obviously reduced, and affinity with pathogenic bacteria can be raised. In the meantime, through inhibiting a QseC acceptor, expression of a key disease-associated gene of a pathogen is inhibited, thus establishing the foundation for the realization of the targeting antibacterial effect of the modifier.

Description

The preparation method and application of daiamid type dendritic macromole structural modification thing

Technical field

The present invention relates to a kind of macromolecular compound, particularly preparation method and the pharmaceutical applications of a kind of daiamid type dendritic macromole structural modification thing, this modifier.

Background technology

At present, the various bacterial resistances that cause due to abuse of antibiotics grow in intensity mondial spreading, and the infectious diseases that many scripts can be cured is becoming the disease that is difficult to healing.The infection of drug-resistant bacteria causes mortality to increase, and infects complication and increases, and resistance pathogenic infection associated diseases has become the common disaster of the universe.Current bacterial resistance has presented to the trend of multidrug resistance development, and some bacteriums have been developed into unmanageable " superbacteria ".In one piece of article of < < lancet > > magazine in August, 2010, introduce a kind of superbug " New Delhi metal-beta-lactamase 1 ", referred to as NDM-1, almost resist all microbiotic, at South Asian nations such as India, occur and spread to American-European a plurality of country, its resistance mechanism resistant organism different from the past is as methicillin resistance bacterium (MRSA), it is a kind of new super drug resistant gene, a kind of new resistance enzyme-metallo-β-lactamase 1 of encoding, can be hydrolyzed nearly all beta-lactam class antibacterials, thereby cause resistance.

At present, mainly contain three major types Resistant strain and threatening the mankind.The first kind is the VRSA of the modal MRSA of comprising and vancomycin resistance.Equations of The Second Kind is multidrug resistance (MDR) Gram-negative bacteria, this bacterioid almost can be resisted available all Antibiotics at present, comprises penicillins, cephalosporins, carbapenems, aminoglycoside, polymyxins, tetracyclines, quinolones, beta-lactam etc.And more difficult for the new antibiotic research and development of Gram-negative bacteria, because its external cell walls can stop most medicine, and its powerful outer row's ability can be discharged cell by unnecessary medicine.The 3rd class is the mycobacterium tuberculosis (MDR-TB and XDR-TB) of multidrug resistance and extensive resistance.This class resistance tubercule bacillus more and more becomes the serious threat of developing country.The treatment of MDR-TB need to reach the treatment course for the treatment of of 2 years, and is attended by very severe side effect; The treatment of XDR-TB is more difficult, and lethality rate is very high.More severe is can resist all antibiotic full drug-resistant bacterias (pan resistant bacteria) to occur, once this bacterioid of human infection may face the condition pasting medical help.In the face of multidrug resistant bacterium is increased and rapid spread increasingly, and the severe situation that after infecting, lethality rate continues to climb to a higher point, how effectively controlling multidrug resistant bacterium infects the harm cause and has become the focus that national governments show great attention to, find and development New Policy and novel drugs that effectively control multidrug resistant bacterium infects, become the goal in research of the world scientist task of top priority.

Microbiotic is that treatment bacterium infects the most classical medicine, yet nearly all microbiotic all can inevitably be induced bacterial resistance when treatment bacterium infects.Since nearly half a century, microbiotic R & D Strategy is still confined to traditional microbiotic structure of modification and modification, and new antibiotic can produce resistance again very soon for clinical rear bacterium, therefore the development of new antibiotic can not fundamentally solve bacterial resistance problem.And the appearance speed of antibiotics resistance bacterial strain is considerably beyond the speed of new antibiotic research and development, clinical effective antibacterials reduce increasingly.In order effectively to resist day by day serious drug-resistant bacteria, must break through conventional thought, find and there are novel texture and not inducibly resistant antibacterials.

Polyamide-amide (polyamidoamine, PAMAM) be a kind of nano level macromolecular cpd of synthetic, due to have monodispersity, internal cavities and surface abundant can modification group etc. unique texture advantage, PAMAM is at biomedicine field, as aspects such as gene drug carriers, solubility promoter, effective catalyst and nano materials, there is application and research very widely.Recently research is found, the aminoterminal PAMAM(PAMAM-NH of high algebraically ₂) externally there is significant anti-microbial activity, as G3.0 and G5.0 can significantly suppress Pseudomonas aeruginosa ( p. aeruginosa) and streptococcus aureus ( s. aureus) growth.Compare with the molecular structure of existing all kinds of microbiotic and antibacterials, PAMAM-NH ₂be the antimicrobial molecule of a class formation novelty, its anti-microbial effect may be relevant with its terminal amino group number.PAMAM-NH ₂synthetic method is ripe, and at present commercially available have tens of kinds of G1.0-G10.0 etc., and along with the increase of algebraically, PAMAM nano-scale, terminal amino group number, shape and constitutional features also can change thereupon.

Although PAMAM-NH ₂be expected to become desirable antibiotic candidate molecules, yet existing literature research is thought, PAMAM has certain cytotoxicity, and toxicity size and algebraically, surface charge, concentration and proportional (the El-Sayed M of incubation time, Ginski M, Rhodes C, Ghandehari H. Transepithelial transport of poly (amidoamine) dendrimers across Caco-2 cell monolayers. j Control Release. 2002; 81 (3): 355-65.); The functional end-group of PAMAM is also to affect its Cytotoxic important factor, and wherein (PAMAM-OH) < of C-terminal is less than the aminoterminal (PAMAM-NH of (PAMAM-COOH) < of C-terminal ₂) (Kitchens KM, El-Sayed ME, Ghandehari H. Transepithelial and endothelial transport of poly (amidoamine) dendrimers. adv Drug Deliv Rev. 2005; 57 (15): 2163-76.).Adopt PAMAM-COOH that serum lactic dehydrogenase detects (LDH) the different algebraically cytotoxicity to Caco-2, found that, showed cell toxicity just when G3.5 and G4.5 PAMAM-COOH concentration are greater than 10mM, and 1mM G3.0 and G4.0 PAMAM-NH ₂cell is produced to overt toxicity (Geiser M, Schurch S, Gehr P. Influence of surface chemistry and topography of particles on their immersion into the lung's surface-lining layer. j Appl Physiol. 2003; 94 (5): 1793-801).

Due to PAMAM-NH ₂cytotoxicity greatly limit its further further investigation and application.Therefore,, how by chemically modified, improve PAMAM-NH ₂target anti-microbial effect, and reduce its cytotoxicity, be a great problem of studying at present.

Summary of the invention

In view of the deficiencies in the prior art, the object of the invention is to by lot of experiments daiamid type dendritic macromole (PAMAM-NH ₂) carry out structural modification research, the daiamid type dendritic macromole structural modification thing that a kind of anti-microbial activity is strong, cytotoxicity is little, preparation method and the pharmaceutical applications of this modifier are provided.

First object of the present invention is achieved in that daiamid type dendritic macromole structural modification thing, and the daiamid type dendritic macromole that is 1:1-3 by mol ratio and formula I or formula II compound pass through amido linkage covalent coupling gained:

。

First object of the present invention can also realize like this: described daiamid type dendritic macromole structural modification thing is 1:1.5-2.3 by mol ratio 3.0 generation daiamid type dendritic macromole pass through amido linkage covalent coupling gained with formula I or formula II compound.

Described daiamid type dendritic macromole structural modification thing, each 3.0 generation daiamid type dendritic macromole end be connected with 1.89 formula II compound molecules.

Second object of the present invention is achieved in that a kind of preparation method of daiamid type dendritic macromole structural modification thing, press the mol ratio of 1:1-3, get daiamid type dendritic macromole and above-mentioned formula I or formula II compound, be dissolved in N, in dinethylformamide, add triethylamine and I-hydroxybenzotriazole, stirring reaction 2-12h, obtains daiamid type dendritic macromole structural modification thing.

Second object of the present invention can also realize like this: the preparation method of described daiamid type dendritic macromole structural modification thing, the consumption mol ratio of triethylamine and daiamid type dendritic macromole is 6-8:1, and the consumption mol ratio of I-hydroxybenzotriazole and daiamid type dendritic macromole is 1-3:1.

The preparation method of described daiamid type dendritic macromole structural modification thing, wherein the consumption mol ratio of daiamid type dendritic macromole and above-mentioned formula I or formula II compound is 1:2.

The preparation method of described daiamid type dendritic macromole structural modification thing, wherein daiamid type dendritic macromole was 3.0 generations.

Because daiamid type dendritic macromole structural modification thing is in its anti-microbial activity of not remarkably influenced, obviously reduced PAMAM-NH ₂cytotoxicity, and can improve the avidity to pathogenic bacteria, by suppressing QseC acceptor, suppress the expression of the crucial Disease-causing gene of pathogenic bacteria simultaneously, for realizing its target anti-microbial effect, lay a good foundation.Therefore, the 3rd object of the present invention is to provide a kind of pharmaceutical applications, i.e. the application of described daiamid type dendritic macromole structural modification thing in preparation antibacterials.Preferably, described antibacterials are target antibacterials.

Compared with prior art, the daiamid type dendritic macromole structural modification thing the present invention relates to has obtained following unforeseeable technique effect: first, this molecule has PAMAM restraining and sterilizing bacteria ability, although surface charge is by LED209 carboxy derivatives partly shielding effect, but very not remarkable on its anti-microbial activity impact, PAMAM-NH ₂after LED209 carboxy derivatives is modified, anti-microbial activity slightly reduces or is several unchanged.Secondly, this molecular cell toxicity is starkly lower than the PAMAM-NH of unmodified ₂.In addition, this molecule has the activity of LED209 molecule, can be used as QseC receptor antagonist, significantly suppresses the expression of Enterohemorrhagic E.coli Disease-causing gene.In addition, this macromolecule water-solubility is good, has target binding ability, synthetic with low cost, is an ideal drug candidate with anti-microbial effect.

Accompanying drawing explanation

Fig. 1 is meta-LED209-PAMAM and PAMAM-NH ₂hydrogen nuclear magnetic resonance spectrogram

Wherein: meta-LED209-PAMAM (A), PAMAM-NH ₂(B).

Fig. 2 is the carbon-13 nmr spectra figure of meta-LED209-PAMAM.

Fig. 3 is the calculation result figure of ultimate analysis.

Fig. 4 is cell survival percentage histogram; Wherein: (A) being cell GES-1, is (B) cell MC 3T3-E1, (C) is cell SW480; Experiment repeats to average for 4 times drafting histogram, and statistical method is tcheck, ^* p<0.05 compares with untreated cell, ^# p<0.05 meta-LED209-PAMAM G3.0 treatment group is compared.

Fig. 5 is LEE1 gene ler, fliC, the horizontal variation diagram of mRNA of flagella genes flhDC and stx2A; Wherein: PCR method detects enterohemorrhagic Escherichia coli EHEC and 50 μ M norepinephrines (NE), 50 μ M meta-LED209-COOH, after 10 μ M or 50 μ M of meta-LED209-PAMAM G3.0 process, LEE1 gene ler, fliC, the mRNA level of flagella genes flhDC and stx2A changes. ^* p<0.05 compares with control group, ^# p<0.05 compares with 50 μ M NE treatment group.

Fig. 6 is the quantitative analysis figure (EHEC) of FITC fluorescence intensity.

Fig. 7 is the quantitative analysis figure (SW480) of FITC fluorescence intensity.

Fig. 8 is the MS collection of illustrative plates of compound 1.

Fig. 9 is the MS collection of illustrative plates of compound 2.

Embodiment

In gram negative pathogenic bacteria QS system, QseC effect is particularly important.QseC is the membranin of 2 membrane spaning domains, belongs to Histidine susceptor kinases, is also bacterium adrenergic and aromatic series signal self-induction agent 3(autoinducer 3 simultaneously, acceptor AI-3).After it is combined with host's adrenergic signaling molecule and AI-3, Qsec starts to start autophosphorylation, triggers again subsequently transcription factor QseB(response regulator) phosphorylation process, finally activate transcribing of crucial Disease-causing gene in bacterium.Therefore, QseC and bacterium pathogenic closely related, becomes desirable antitoxin power drug targets.LED209 is a kind of potent and avirulent QseC inhibitor, and the verified QseC that can effectively suppress is combined with signaling molecule, and then suppresses the expression of downstream Disease-causing gene, reduces the pathogenic of bacterium.The structural formula of LED209 is as follows:

。

The present invention passes through PAMAM-NH ₂as main body, take LED209 carboxy derivatives as coupling group, by covalently bound synthesizing polyamides amine type dendritic macromole structural modification thing, obtained having the novel cpd of bacterium target and difunctional anti-microbial effect.Below that LED209 carboxy derivatives and PAMAM-LED209 are synthetic, effect embodiment.

synthesizing of embodiment 1:LED209 carboxy derivatives

Be below the synthetic route of two kinds of LED209 carboxy derivatives, be respectively contraposition (para-) and a position (meta-) carboxyl, synthetic route is as follows:

。

(1) compound 1 is synthetic: get 100 mL round-bottomed flasks, add methylene dichloride (DCM, 50 mL), in the time of stirring, add successively aniline (SM-2,5.1 g, 54.8 mmol), triethylamine (11g, 109 mmol) and p-acetaminobenzenesulfonyl chloride (SM-1,11.68 g, 50 mmol), stirring at room 10 h.Inferior daily 100 mL DCM diluting reaction solution, add 1 N HCl solution 30 mL washing organic phase 3 times, use Na ₂sO ₄after finish-drying, be spin-dried for, can obtain target compound 12 g, productive rate 82.7%.Compound 1 theoretical molecular 290.5 detected value M-1 peaks are that 289.45(is shown in Fig. 8).

(2) synthesizing of compound 2: to compound 1(12 g), add 1 N HCl (50 mL), stirring and refluxing 12 h.Add next day appropriate sodium bicarbonate to be neutralized to pH7.0, with ethyl acetate (EA) extractive reaction liquid, Na ₂sO ₄dry organic phase, is spin-dried for, and can obtain target compound 8 g, productive rate 78%.It is that 247.4(is shown in Fig. 9 that intermediate compound 2 theoretical moleculars 248.5 detect M-1 peak).

(3) compound 3 is synthetic: accurately take compound 2(496 mg) be dissolved in acetone (10 mL), then add 4-carboxyl thiocarbanil (SM-3,358 mg), reaction soln stirring and refluxing 24 h.Filtering reacting solution obtains the faint yellow compound crude product of target, through acetone recrystallization, obtains target compound LED209 contraposition carboxy derivatives (para-LED209-COOH) white powder 420 mg, productive rate 49.2%.

NMR measures on MERCURY-PLUS 400 and AVANCF300MHZ, take D6-DMSO as solvent, take tetramethylsilane TMS as interior mark, observing frequency is 300 MHz.

HPLC chromatographic condition: acetonitrile/water=3/7(is containing 0.05% trifluoroacetic acid), Ven μ sil XBP-C ₁₈(5 μ m, 4.6 * 250 mm); Ultraviolet detection wavelength 254 nm; Flow velocity 1 mL/min; 30 ℃ of column temperatures; 25 ℃ of sample temperatures; Sample size: 20 μ L.

Mass spectrometry results: MS(ESI), [M+1] ⁺ m/z428.1, substantially conform to theoretical value 427.5; ¹h NMR (D6-DMSO, 300 MHz) result: δ: 12.79-12.78 (d, 1H ,-COO h), 10.37-10.25 (t, 3H ,-N h-), 7.90-7.62 (m, 2H ,-Ph), 7.26-7.21 (m, 6H ,-Ph), 7.09-6.99 (m, 5H ,-Ph), show that hydrogen ownership is errorless with synthesized target product structure matching, further confirm successfully synthetic para-LED209-COOH.Purity detecting result: HPLC shows that para-LED209-COOH purity is greater than 95%.

(4) compound 4 is synthetic: accurately take compound 2(496 mg) be dissolved in acetone (10 mL), then add 3-carboxyl thiocarbanil (SM-4,358 mg), reaction return stirring one week.After reaction finishes, filtering reacting solution obtains target compound crude product gray solid powder 420 mg, productive rate 49.2%.Then reaction soln, through column chromatography for separation (DCM:MeOH=20:1), obtains carboxy derivatives (meta-LED209-COOH) pale yellow powder 150 mg in position between target compound LED209, productive rate 17.5%.

NMR measures on MERCURY-PLUS 400 and AVANCF300MHZ, take D6-DMSO as solvent, take tetramethylsilane TMS as interior mark, observing frequency is 300 MHz.

HPLC chromatographic condition: acetonitrile/water=3/7(is containing 0.05% trifluoroacetic acid), Ven μ sil XBP-C ₁₈(5 μ m, 4.6 * 250 mm); Ultraviolet detection wavelength 254 nm; Flow velocity 1 mL/min; 30 ℃ of column temperatures; 25 ℃ of sample temperatures; Sample size: 20 μ L.

Mass spectrometry results: MS(ESI), [M+1] ⁺ m/z428.1, substantially conform to theoretical value 427.5; ¹h NMR (D6-DMSO, 300 MHz) result: δ: 10.47-10.43 (m, 2H ,-N h-), 10.27-10.26 (t, 1H ,-N h-), 8.09 (s, 2H ,-Ph), 7.76-7.74 (s, 2H ,-Ph), 7.67-7.64 (m, 2H ,-Ph), 7.50-7.45 (m, 2H,-Ph), 7.29-7.23 (m, 4H,-Ph), 7.07-7.02 (m, 1H,-Ph), show that hydrogen ownership is errorless with synthesized target product structure matching, further confirm successfully synthetic meta-LED209-COOH.Purity detecting result: HPLC shows that meta-LED209-COOH purity is greater than 95%.

embodiment 2: para/meta-PAMAM G3.0-LED209's is synthetic

Precision takes the para-LED209-COOH(30 mg of embodiment 1 preparation, 0.070 mmol), G3.0 PAMAM-NH ₂(242 mg, 0.035 mmol) is dissolved in DMF (DMF, 10 mL), adds triethylamine (21 mg, 0.21 mmol) and I-hydroxybenzotriazole (HOBT, 9.5 mg, 0.070 mmol), reaction soln stirred overnight at room temperature.Reaction soln, through preparative chromatography purifying (pre-HPLC), obtains faint yellow oily matter 55 mg.

Precision takes meta-LED209-COOH(30 mg of embodiment 1 preparation, 0.070 mmol), G3.0 PAMAM-NH ₂(242 mg, 0.035 mmol) is dissolved in DMF (DMF, 10 mL), adds triethylamine (21 mg, 0.21 mmol) and I-hydroxybenzotriazole (HOBT, 9.5 mg, 0.070 mmol), reaction soln stirred overnight at room temperature.Reaction soln, through preparative chromatography purifying (pre-HPLC), obtains faint yellow oily matter 56mg.NMR measures on AVANCF300MHZ, with D ₂o is solvent, take TMS as interior mark.

The G3.0 PAMAM proton nmr spectra of unmodified the results are shown in Figure 1(B): ¹h NMR (D ₂o, 400 MHz) δ: 4.813 (D ₂o, residual solvent peak), 3.30-3.25 (C h ₂c h ₂nH ₂), 2.832 (CON h), 2.728 (CH ₂nH ₂), 2.637 (N h ₂), 2.438 (CH ₂cON h); Position between connection LED209() after carboxy derivatives, ¹h NMR collection of illustrative plates generation considerable change, see Fig. 1 (A): LED209 goes out peak position δ: 8.59,8.25 and 7.47(LED209 phenyl ring hydrogen), show LED209-COOH(meta-) be successfully connected to PAMAM G3.0.In addition, meta-PAMAM G3.0-LED209's that prepared by the present embodiment ¹³c NMR (D6-DMSO, 100 MHz) the results are shown in Figure all C chemical shifts of 2:PAMAM G3.0 all between 52-30, so contriver judges that δ 49.41 should belong to PAMAM skeleton carbon, and δ 41.16-39.49 should belong to DMSO solvent peak.δ 149.96,140.14, and 135.16,128.68,120.47 belong to LED209 phenyl ring carbon goes out peak.From ¹h NMR and ¹³c NMR collection of illustrative plates result, can affirm, LED209-COOH(meta-) is successfully connected with PAMAM G3.0, forms a new compound.

For further proving whether LED209 reacts according to design mol ratio (2:1) with PAMAM, and we have done ultimate analysis.G3.0 PAMAM (molecular formula C ₃₀₂h ₆₀₈o ₆₀n ₁₂₂, 6909) results of elemental analyses in Table 1.According to detected result and the calculated results, it is X-coordinate that the PAMAM end of take is substituted number (the LED209 number of each PAMAM minute sub-connection), take each element theory content value is ordinate zou, draw coordinate diagram curve, then according to the actual detected value of PAMAM-LED209, from corresponding figures, find PAMAM end to be substituted number.The results are shown in Figure 3, according to the content of carbon, hydrogen, nitrogen and sulphur, calculate respectively that may to replace number be 1.7,2.0,1.75 and 2.1, mean value is 1.89, and products therefrom is meta-LED209-PAMAM _(n=1.89).

The results of elemental analyses of table 1 G3.0 PAMAM

Note: △ is the difference of PAMAM G3.0 and PAMAM-LED209.

embodiment 3:G3.0 PAMAM-LED209 antibacterial activity in vitro and cytotoxicity are investigated

1, material and instrument

1.1 experimental strain

12 strain Gram-negative bacterias comprise that 9 strain reference cultures and 3 strain Resistant strain (in Table 2) test for this part.

9 strain reference cultures and 3 strain Resistant strain that table 2 test is used

Chinese	English name	English abbreviation
			Reference culture	Type strain	TS
Intestinal bacteria	Escherichia coli ATCC 25922	E. coli
			Intestinal bacteria	Escherichia coli K12MG1655	E. coli MG 1655
Pseudomonas aeruginosa	261H Pseudomonas aeruginosa ATCC 27853	P. aeruginosa
			Klebsiella pneumoniae	Klebsiella pneumonia ATCC13883	K. pneumonia
Salmonella enteritidis	262H Salmonella enterica ATCC 9150	S. enteric
			Acinetobacter bauamnnii	Acinetobacter baumanni	A. baumanni
Shigella flexneri	Shigella flexneri	S. flexneri
			Salmonella typhimurium	Salmonella typhimurium	S. typhimurium
Enterohemorrhagic Escherichia coli	Entero-hemorrhagic E.coli O157:H7	EHEC
			Resistant strain	Drug resistant strain	MDR
Multidrug resistant intestinal bacteria	Multi-drug resistant E. coli XJ74283	MDR-EC
			Produce the klebsiella pneumoniae of ESBLs	ESBLs-producing K. pneumonia XJ75297	ESBLs-KP
Produce the intestinal bacteria of ESBLs	ESBLs-producing E. coli ATCC 35218	ESBLs-EC

1.2 experiment clone used

In experiment, clone used has CCL188 SW480, and people's gastric mucosal cell is the former scleroblast MC3T3-E1 of GES-1 and mouse.

1.3 reagent, test kit and medicine

N,O-Diacetylmuramidase, tetrazolium bromide (3-(4,5-dimethylthiazole-2)-2,5-phenylbenzene tetrazole bromine salt, MTT), bovine serum albumin (BSA), EDTA and Tris be purchased from Aldrich-Sigma company; Foetal calf serum (FBS), DMEM high glucose medium and trypsinase are purchased from U.S. Gibico company; The analytical reagent such as acetic acid, dehydrated alcohol, glycerine, paraffin, dimethyl sulfoxide (DMSO), dimethylbenzene and formaldehyde are purchased from Tianjin reagent company.

Benzylpenicillin sodium for injection (80Wan unit) is purchased from Zhong Nuo pharmaceutcal corporation, Ltd; Streptomycin sulphate for injection (100Wan unit) is purchased from Dalian Metro medicine company; Microbiotic Ampicillin Trihydrate, Oxazacillin, levofloxacin, ceftazime, erythromycin are purchased from Nat'l Pharmaceutical & Biological Products Control Institute.

G3.0 PAMAM-LED209: the embodiment of the present invention 2 is prepared gained meta-LED209-PAMAM _(n=1.89) .

RNAprep pure culturing cell/bacterium total RNA extraction reagent box is purchased from Tian Gen biochemical technology company limited; 260HSYBR (R) PrimeScriptTM RT-PCR Kit and PCR related reagent are purchased from precious biotechnology company limited; Fluorescein isothiocyanate FITC(Sigma company); Instant dialysis tubing (MWCO:2000 Da) is purchased from upper sea base star bio tech ltd; Other chemical reagent used are analytical pure.

1.4 main solution formulas

m-H broth culture:2.5 g M-H meat soup dry powder, add 125 μ L MgCl ₂solution is (containing Mg ²⁺10 mg/mL) and 200 μ L CaCl ₂solution is (containing Ca ²⁺10 mg/mL), then with deionized water, be settled to 100 mL, 121 ℃ of autoclavings, 20 min.

M-H nutrient agar: 3.8 g M-H nutrient agar dry powder, add 100 mL distilled water to mix, standing 10 min, after dry powder dissolves completely, 121 ℃ of autoclavings, 20 min.Then spread M-H nutrient agar 48 ℃ time dull and stereotyped.

Nutrient broth medium: 1.9 g nutrient broth dry powder, add 100 mL cold distilled waters to mix, standing 10 min, low baking temperature boils (every asbestos wire net or magnetic force heating stirrer), after dry powder dissolves completely, 121 ℃ of autoclavings, 20 min.

0.5 Maxwell is than turbid liquid: at 0.18 mol/L H ₂sO ₄(1%, v/v) in 99.5 mL, add 0.048 mol/L BaCl ₂(1.175%, w/v) 0.5 mL, after fully mixing, its turbidity is 0.5 Maxwell than turbid standard, is equivalent to 5 * (10 ⁷-10 ⁸) CFU/mL.Keeping in Dark Place in dark place, in half a year, uses, and should fully mix before use.

PBS damping fluid; Accurately weigh KCl 0.2 g, KH ₂pO ₄0.24 g, NaCl 8.0 g, Na ₂hPO ₄2H ₂o 1.56 g, after adding 100 mL deionized waters fully to dissolve, adjust pH to 7.4, packing after constant volume 100 mL, and 121 ℃ of autoclavings, 20 min, are 0.1M PBS mother liquor.It,, with 10 times of sterilized water dilutions, is to 0.01M PBS solution, matching while using.

Without RNA enzyme aqua sterilisa: after the in-built 999 mL distilled water of the vial that high bake is crossed (180 ℃, 8 h), add the DEPC of 1 mL, fully mix, normal temperature is placed after 12 h 121 ℃ of autoclavings, and 20 min, place refrigerator standby.

N,O-Diacetylmuramidase (400 μ g/mL): take N,O-Diacetylmuramidase standard sterling, with 0.01M PBS, be formulated as 1000 μ g/mL stostes, and dilution be 400 μ g/mL reference liquids, after packing ,-20 ℃ of Refrigerator stores are standby.

50 * TAE damping fluid: take respectively Tris 242 g, Na ₂eDTA2H ₂o 37.2 g are added in 1 L beaker, then add approximately 800 mL deionized waters, stir, then add the glacial acetic acid of 57.1 mL, after fully dissolving, add deionized water and are settled to 1 L, room temperature preservation.

1.0% agarose: 1 g agar powder is dissolved in 100 mL 1 * TAE damping fluids, is heated to melt completely in microwave oven, adds the ethidium bromide solution 50 μ L of 1 mg/mL after slightly cool, fully mixes rear paving glue.

2, method

The mensuration of 2.1 minimum inhibitory concentrations (MIC)

2.1.1 MIC measuring method

A. collecting cells: get frozen in the bacterial strain to be measured of 20 ℃ of ﹣, streak inoculation is in M-H agar plate, overnight incubation in 37 ℃ of incubators, inferior daily transfering loop picking mono-clonal bacterium colony, is inoculated in 3 mL nutrient broths 37 ℃, 220 r/min, rise in value to logarithmic growth after date, with M-H meat soup, bacterium liquid is diluted to 0.5 Maxwell than turbid standard, approximately 1 * 10 ⁸cFU/mL.Then use M-H meat soup by standby after the dilution in 1: 100 of above-mentioned bacteria suspension.Bacterium liquid after dilution should be inoculated in 15 min.

B. the dilution of antibacterials and the inoculation of bacterium liquid: G3.0 PAMAM-LED209 is diluted to 400 μ g/mL with sterilized water, filtration sterilization, packing is standby.Ampicillin Trihydrate 0.1M, the phosphate buffered saline buffer dilution of pH 6.0), Oxazacillin, Ciprofloxacin aseptic deionized water, the sterile sodium carbonate aqueous solution for ceftazime (anhydrous sodium carbonate be ceftazime used 10%), aseptic deionized water for levofloxacin (0.1 M NaOH hydrotropy), be diluted to respectively 1024 μ g/mL, 20 ℃ of Refrigerator stores of ﹣ are standby.

C. application of sample: get 10 row=60 holes, row * 6 at the aseptic 96 non-edges of orifice plate, every row is labeled as respectively " G3.0 PAMAM-LED209, microbiotic contrast, not containing the growth control of medicine ".Every hole adds respectively 100 μ L M-H meat soups, and every row adds 100 μ L G3.0 PAMAM-LED209, microbiotic, M-H meat soup successively according to flag sequence in the 1st row hole.After fully mixing with the volley of rifle fire, from every hole sucking-off 100 μ L, join the 2nd corresponding hole, the like, 2 times of doubling dilutions to the 10 are listed as, and discard 100 μ L liquid of last sucking-off.Then to add the bacteria suspension to be measured of the above-mentioned preparation of 100 μ L to make the whole bacterial concentration of every Kongzui be 5 * 10 in every hole ⁵cFU/mL.In the periphery of 96 orifice plates, add 200 μ L bacteria suspension to be measured (not containing medicine) as aseptic contrast.Attention should be got bacteria suspension to be measured simultaneously and be uploaded culture at agar plate, to examine bacteria-checking liquid purity.

D. hatch with result and judge: by shaking 1 min on the 96 orifice plate micro oscillators of having inoculated, fully mixing, be placed in wet box, in 37 ℃ of incubators, hatch 16 ~ 20 h, to the staphylococcus of methicillin-resistant and vancomycin resistance, should continue to hatch 24 h.With the lowest drug concentration in the limpid hole of the visual inspection MIC of medicine for this reason, whether the bacterial growth situation that simultaneously should check growth control hole is good, whether aseptic control wells is limpid, the flat board that checks bacteria suspension is cultivated situation to determine whether it pollutes, and whether the MIC value of Quality Control bacterial strain is in Quality Control scope.This measures and repeats 3 times.

The cytotoxicity of 2.2 G3.0 meta-LED209-PAMAM

Experiment adopts MTT method, detect the cytotoxicity of G3.0 meta-LED209-PAMAM to MC3T3, GES-1 and SW480, specific experiment method is as follows: testing cell used is GES-1(people's gastric epithelial immortal cell line, by Xijing hospital of The Fourth Military Medical University, digesting doctor Chai Na of disease hospital friendship presents), MTT laboratory reference literature method, specific as follows: with the GES-1 cell of increased logarithmic phase, with the DMEM substratum that contains 10% foetal calf serum, to prepare 2.5 * 10 ⁴cells/mL single cell suspension, every hole 200 μ L are inoculated in 96 holes, after 48 h, discard substratum, with 0.01 M PBS drip washing cell 3 times, then with containing the DMEM substratum of serum, 37 ℃ of hungry cultivations after 4 h, discard substratum, add the medicine to be measured of different concns, hatch 48 h, carry out MTT detection, detect wavelength 490 nm.Take not processed cell enzyme activity as 100%, calculate the cytoactive that drug treating is crossed.Calculation formula: cell viability (%)=(OD ₁-OD ₂)/(OD ₃-OD ₄), OD wherein ₁refer to the cell OD value that treated with medicaments is crossed, OD ₂refer to the OD value that does not have cell only to have medicine, OD ₃refer to untreated cell OD value, OD ₄refer to the acellular OD value of blank well during without medicine.

2.3 pairs of regulation and control that EHEC Disease-causing gene is expressed

By PCR method, detect the impact that G3.0 meta-LED209-PAMAM expresses EHEC Disease-causing gene, method is as follows:

A) bacterium liquid is prepared: get frozen in the bacterial strain EHEC to be measured of 20 ℃ of ﹣, streak inoculation is in M-H agar plate, overnight incubation in 37 ℃ of incubators, inferior daily transfering loop picking mono-clonal bacterium colony, is inoculated in 3 mL nutrient broths 37 ℃, 220 r/min, rise in value to logarithmic growth after date, with DMEM substratum, bacterium liquid is diluted to 0.5 Maxwell than turbid standard, approximately 1 * 10 ⁸cFU/mL.

B) the dilution of para/meta-LED209-COOH:precision takes contraposition or a position carboxyl LED209, with DMSO, dissolves preparation 50 mM mother liquors, and then with sterilized water stepwise dilution to testing concentration used.

C) norepinephrine preparation:precision takes norepinephrine (NE), with appropriate sterilized water, dissolves preparation 500 mM mother liquors, filtration sterilization, ℃ preservation of packing lucifuge-20.

D) application of sample and sample collection:get aseptic 96 orifice plates, 6 row * 6 row in the middle of getting, every hole adds 180 μ L bacterium liquid, then the 1st row adds 20 μ L DMEM, all the other every holes add 10 μ L NE(1000 μ M), then from the 2nd, walk to the 6th row and add respectively successively 10 μ L DMEM, 10 μ L meta-LED209-COOH(20 μ M), 10 μ L meta-LED209-COOH(1000 μ M), 10 μ L para-LED209-COOH(20 μ M), 10 μ L para-LED209-COOH(1000 μ M), after fully mixing, lucifuge is put in cultivation 5 h in 37 ℃ of wet boxes.Then collect every row bacterium liquid in aseptic centrifuge tube, freezing preservation.

E) the total RNA of bacterium extracts: according to sky root test kit specification sheets, strictly operate, after having extracted, survey concentration and the purity of total RNA with DU800 nucleic acid-protein analyser.

F) rTreference reagent box specification sheets, testing reaction system used is 20 μ L, wherein 5 * Primer

Script Buffer 4 μ L, RT Enzyme Mix I 1 μ L, OligodT primers 1 μ L, Random 6 mers 1 μ L, RNA10 μ L, RNase free dH ₂o supplies 20 μ L.37 ℃ of reaction conditionss, 15 min, 85 ℃, 5 s, 4 ℃, 10 min.After reaction finishes, cDNA is put in to-20 ℃ of preservations.

G) sxemiquantitative PCR reaction system is 25 μ L, cDNA 2 μ L wherein, positive each 1 μ L of anti-primer, 2 * Taq Mix, 12.5 μ L, ddH ₂o supplies 25 μ L.Reaction conditions: 94 ℃ of denaturation 5 min, 94 ℃ of sex change 30 s, 58 ℃ of annealing 30 s, 72 ℃ are extended 45 s, read plate, 30 circulations.Experiment the primer sequence and corresponding annealing temperature are shown in Table 3-1.

Table 3 experiment the primer sequence and corresponding annealing temperature

2.4 fluorescence microscope

2.4.1 the preparation of cell climbing sheet

A) slide is coated:slide (1 cm * 1 cm) is cleaned to post-drying, be soaked in the vitriol oil 24 h completely, then use tap water cleaning down 20 times, then after drying for 10 times with deionized water rinsing, be placed on 121 ℃ of clean lunch box autoclave sterilizations, 20 min, standby.Then according to experimental design, take out aseptic slide and be put in culture dish, add 0.01% appropriate poly-lysine, placement is spent the night.Before use, abandon supernatant next day, and clean 3 times with aseptic PBS, dries standby.

B) cell climbing sheet:by stand-by cell, with after trysinization, prepare single cell suspension 5 * 10 ⁴/ mL, then inoculates in culture dish (each culture dish contains 4 slides).Note, a small amount of substratum is dripped in the position of first preparing to put slide before inoculation in culture dish, and slide and culture dish are bonded together, and slide levitating while preventing from adding cell, causes double-layer cell adherent.In whole process, want aseptic technique in addition.

C) dosing is processed:after cell attachment, according to experimental design, culture dish is labeled as respectively to " contrast, G3.0-FITC and G3.0-LED209-FITC ".First abandon substratum supernatant, with PBS, clean cell 1 time, then add successively respectively " it is 20 μ M that the G3.0-FITC(of serum free medium, serum free medium preparation makes its final concentration) and G3.0-LED209-FITC(to make its final concentration be 20 μ M) ", lucifuge continue to be cultivated, and respectively at 1,3,5, during 9 h, gets 1 of slide, after cleaning with PBS, add fixedly 15-20 min of 4% paraformaldehyde, and then with PBS, clean 3 times fluorescence microscope.Whole process is noted lucifuge operation.

2.4.2 the preparation of bacterium creep plate

A) the preparation of bacterium liquid:prepare fresh EHEC bacteria suspension, method is with experiment third part 2.5.2.

B) experimental design:get 96 orifice plate three row, be labeled as respectively " contrast, G3.0-FITC and

G3.0-LED209-FITC ", then every hole adds EHEC bacterium liquid 180 μ L(1 * 10 ⁸cFU/mL), then in corresponding row, add " sterilized water, G3.0-FITC(200 μ M) and G3.0-LED209-FITC(200 μ M) " each 20 μ L, 37 ℃ of cultivations, in different point in time sampling, remove supernatant after centrifugal, with PBS repeated washing three times, add 3% glutaraldehyde 50 μ L, drop to clean slide, after lucifuge is air-dry, fluorescence microscope.

2.4.3 every slice, thin piece of fluorescent quantitation is chosen 5 visuals field, take same exposure parameter to take pictures, to adopted slice, thin piece fluorescent quantitation, the fluorescent quantitation method of every pictures: adopt the fluorescent quantitation of Image Pro plus computed in software pictures taken, method is: fluorescence area x average fluorescent strength.Experiment is averaged in triplicate, using mean value as the index of weighing medicine and bacterium or cytosis.

3, result

3.1 G3.0 meta-LED209-PAMAM lowest bacteria fogging-resistant concentration determinings

The minimum inhibitory concentration of table 4 PAMAM and meta-LED209-PAMAM

note.cAZ, ceftazidime; MDR, multi-drug resistance; ESBLs, extended-spectrum β-lactamase; KP, klebsiella pneumoniae; a.baumanni), acinetobacter baumanni..

Contriver has detected altogether 12 strain Gram-negative bacterias and has comprised 9 strain reference cultures and 3 strain Resistant strain, the results are shown in Table 4.Select e.coliaTCC 25922 is as Quality Control bacterial strain, and according to CLSI regulation, ceftazime is 0.06-0.5 μ g/mL to the MIC standard range of ATCC25922.It is 0.5 μ g/mL to the MIC value of ATCC25922 that experiment records ceftazime, meets the requirements, and illustrative experiment method accurately and reliably.Meta-LED209-PAMAM _(n=1.89)the bacterial strain of surveying ( s.typhimuriumexcept ESBLs-KP) all show very strong bacteriostatic action, compare with the PAMAM G3.0 of unmodified (6.25-12.5 μ g/mL), MIC value is constant or raise 1 times (12.5-25 μ g/mL), this prompting, low replacement part is modified PAMAM G3.0, itself anti-microbial activity of not remarkably influenced.

3.2 G3.0 meta-LED209-PAMAM cytotoxicities

MTT results suggest, G3.0 PAMAM-NH ₂with LED209-PAMAM, the cytotoxicity of GES-1, SW480 and MC3T3 is to concentration dependent (see figure 4).When drug level is less than or equal to 102.4 μ g/mL, PAMAM-NH ₂with LED209-PAMAM, the toxicity of GES-1 cell is not had to significant difference, but when drug level reaches 512-1024 μ g/mL, LED209-PAMAM treatment group cell survival rate (87.12%-78.66%) do not have significant difference with cellular control unit; And the PAMAM treatment group cells survival rate (64.58%-60.48%) of unmodified obviously reduces, compared with control group significant difference ( p<0.05), compare with LED209-PAMAM treatment group and also have significant difference ( p<0.05) (Fig. 4 (A)).Comprehensively compare with first part experimental result, meta-LED209-PAMAM after modification is suitable with G2.0 PAMAM cytotoxicity to the cytotoxicity of GES-1, this and our expected results are in full accord, and PAMAM G3.0 surface amino groups can reduce its cytotoxicity after partly modifying.

, from Fig. 4 (B) and Fig. 4 (C), can find out, compare with PAMAM G3.0, the cytotoxicity of the PAMAM-LED209 after modification obviously reduces meanwhile, when high density 512 and 1024 μ g/mL cell survival rate have significant difference ( p<0.05).The cytotoxicity detected result that comprehensive three strains need not be originated, we can find out, the cytotoxicity of PAMAM and PAMAM-LED209 is relevant to concentration and cell category, and the latter's cytotoxicity is significantly lower than the former, this further points out, and we design synthetic PAMAM-LED209 is a potential safe antibiotic candidate molecules.

The impact that 3.3 G3.0 meta-LED209-PAMAM express EHEC Disease-causing gene

Meta-LED209-COOH is connected to PAMAM macromole surface, whether has affected the combination with bacterium QseC acceptor, and we have further detected meta-LED209-PAMAM by PCR _(n=1.89)impact on EHEC Disease-causing gene in the expression of mRNA level.As shown in Figure 5, first 50 μ M NE activate EHEC mycoderm surface QseC acceptor and impel QseC to start autophosphorylation result, then trigger the phosphorylation process of transcription factor QseB, finally activate the crucial Disease-causing gene of EHEC stx2A, flhD, fliCwith lertranscript and expression, results suggest 50 μ M NE have significantly improved each mRNA level that detects gene, to compared with untreated fish group significant difference ( p<0.05).50 μ M and 10 μ M meta-LED209-PAMAM _(n=1.89)all can significantly suppress EHEC Disease-causing gene in the expression of mRNA level, with 50 μ M NE group contrasts have significant difference ( p<0.05), still compare no difference of science of statistics with 50 μ M meta-LED209-COOH groups.Result further confirms, PAMAM macromolecular structure does not affect the avidity of LED209 and QseC acceptor, still has higher intrinsic activity, 10 μ M meta-LED209-PAMAM _(n=1.89)can significantly suppress the expression of the crucial Disease-causing gene in EHEC downstream, reduce the pathogenic of bacterium.

3.4 fluorescence microscope

In order further to confirm the difference of G3.0 PAMAM-LED209 and bacterium and cytosis, the G3.0 and the G3.0-LED209 that are marked with FITC are hatched altogether with bacterium EHEC, cell SW480 respectively, in different time point fluorescence microscopes.Found that, along with the prolongation of time, the fluorescence intensity of EHEC obviously strengthens, and when quantitative fluorescence analysis result confirms G3.0-LED209 and EHEC effect 1,3 and 5 h, fluorescence intensity is significantly higher than G3.0( p<0.05), the association rate of this prompting G3.0-LED209 and EHEC and ability, apparently higher than the G3.0 of unmodified, confirm that this novel molecular has potential targeting.

SW480 cell fluorescence microscope found that, along with time lengthening, cell fluorescence intensity also obviously strengthens; But G3.0 and G3.0-LED209, put and in 1-5 h, almost not to find fluorescent signal in experiment well below EHEC the avidity of SW480 detection time; Quantitative fluorescence analysis result shows simultaneously, point at one time, the G3.0 modifying with LED209 to the avidity of cell also lower than the G3.0 of unmodified, the G3.0 treatment group cell fluorescence intensity that while acting on 15 h, LED209 modifies is significantly lower than G3.0 group (see figure 7), and what G3.0-LED209 cytotoxicity this also can further be interpreted as lower than G3.0.Therefore, the fluorescence microscope result of comprehensive bacterium and cell, can confirm that G3.0-LED209 has improved the avidity to bacterium EHEC, has the antibiotic effect of target.

4, discuss

LED209 be one strong effectively and the QseC acceptor inhibitor of safety and low toxicity, as far back as 2008, be found.But this molecule utmost point is insoluble in water, oral administration bioavailability is lower.The meta-LED209-PAMAM that we are synthetic _(n=1.89)complete water soluble, this be because, in PAMAM molecule, hydrocarbon chain is lipophilicity group, and N-terminal is hydrophilic radical, therefore itself there is the effect that solubilising, breakdown of emulsion and the tensio-active agent such as stable have, even by electrostatic interaction and LED209 formation mixture, can increase equally the solvability of LED209.We are by new couplings meta-LED209-PAMAM of covalently bound formation _(n=1.89), with PAMAM similar, therefore still there is the effect of tensio-active agent, obviously improved the solvability of LED209.

The compound meta-LED209-PAMAM that this is brand-new _(n=1.89)as antibacterials, there is following advantage: first, this molecule has PAMAM restraining and sterilizing bacteria ability, although surface charge is by LED209 partly shielding effect, but it is not very remarkable on its anti-microbial activity impact, PAMAM G3.0 is after LED209 modifies, and anti-microbial activity slightly reduces or be several unchanged, this be because, on average each PAMAM molecule, only connected 1.89 LED209 molecules, less to the electric charge number of its end and Effects of Density.Secondly, this molecular cell toxicity is starkly lower than the PAMAM G3.0 of unmodified.In addition, this molecule has the activity of LED209 molecule, can be used as QseC receptor antagonist, significantly suppresses the expression of Enterohemorrhagic E.coli Disease-causing gene.In addition, this macromolecule water-solubility is good, synthetic with low cost, is an ideal drug candidate with target anti-microbial effect.The result of fluorescent microscope confirms that the PAMAM G3.0 that LED209 modifies has significantly improved the target binding ability to EHEC, has reduced the nonspecific action to cell SW480.This results suggest, meta-LED209-PAMAM _(n=1.89)there is the antibiotic function of potential target.

Claims (8)

1. daiamid type dendritic macromole structural modification thing, is characterized in that: by mol ratio, be 1:1-3 3.0 generation daiamid type dendritic macromole and formula I or formula II compound by amido linkage covalent coupling gained:

2. daiamid type dendritic macromole structural modification thing according to claim 1, is characterized in that: by mol ratio, be 1:1.5-2.3 3.0 generation daiamid type dendritic macromole and formula I or formula II compound by amido linkage covalent coupling gained.

3. daiamid type dendritic macromole structural modification thing according to claim 2, is characterized in that: each 3.0 generation daiamid type dendritic macromole end be connected with 1.89 formula II compound molecules.

4. the preparation method of daiamid type dendritic macromole structural modification thing described in a claim 1, it is characterized in that: the mol ratio of pressing 1:1-3, get 3.0 generation daiamid type dendritic macromole and formula I or formula II compound, be dissolved in N, in dinethylformamide, add triethylamine and I-hydroxybenzotriazole, stirring reaction 2-12h, obtains daiamid type dendritic macromole structural modification thing.

5. the preparation method of daiamid type dendritic macromole structural modification thing according to claim 4, it is characterized in that: triethylamine and 3.0 generation daiamid type dendritic macromole consumption mol ratio be 6-8:1, I-hydroxybenzotriazole and 3.0 generation daiamid type dendritic macromole consumption mol ratio be 1-3:1.

6. according to the preparation method of daiamid type dendritic macromole structural modification thing described in claim 4 or 5, it is characterized in that: 3.0 generation daiamid type dendritic macromole and the consumption mol ratio of formula I or formula II compound be 1:2.

7. the application of daiamid type dendritic macromole structural modification thing in preparation antibacterials described in claim 1-3 any one.

8. the application of daiamid type dendritic macromole structural modification thing in preparation antibacterials according to claim 7, is characterized in that: described antibacterials are target antibacterials.

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