A brain-vectored angiopep-2 based

A brain-vectored angiopep-2based polymeric micelles for the treatment of intracranial fungal infection

Kun Shao a ,Jiqin Wu b ,Zhongqing Chen c ,Shixian Huang a ,Jianfeng Li a ,Liya Ye d ,Jinning Lou d ,Liping Zhu b ,**,Chen Jiang a ,*

Key Laboratory of Smart Drug Delivery,Ministry of Education &PLA,Department of Pharmaceutics,School of Pharmacy,Fudan University,826Zhangheng Road,Shanghai 201203,China b

Department of Infectious Diseases,Huashan Hospital,Fudan University,Shanghai 200040,China c

Department of Pathology,Huashan Hospital,Fudan University,Shanghai 200040,China d

Institute of Clinical Medical Sciences,China-Japan Friendship Hospital,The Ministry of Health,2East Yinghua Road,Beijing 100029,China

a r t i c l e i n f o

Article history:

Received 15May 2012Accepted 22June 2012

Available online 11July 2012Keywords:

Blood-brain barrier Intracranial infection Angiopep-2Amphotericin B Polymeric micelles P-gp

a b s t r a c t

One of the most common life-threatening infections in immunosuppressive patients,like AIDs patients,is cryptococcal meningitis or meningoencephalitis.Current therapeutic options are mostly ineffective and mortality rates remain high.Hydrophobic antifungal drug Amphotericin B (AmB),has become a golden standard in severe systemic fungal infection therapy.However,most AmB commercial formulations,including deoxycholate AmB and lipid formulations of AmB,show poor penetration into the CNS and dif ?culty to reach the therapeutic levels.To improve the CNS permeability of AmB,we have successfully developed an effective brain-targeting polymeric micellar system with angiopep-2modi ?ed,named Angiopep-PEG-PE/AmB polymeric micelles.An immunosuppressive murine model with Crypto-coccus neoformans meningoencephalitis (CNME)was established to evaluate the CNS penetration ef ?-ciency and antifungal treatment ef ?cacy of the AmB-incorporated brain-vectored polymeric micellar formulation,compared with the AmB commercial formulations.After three consecutive days of i.v.administration,the results showed that the group treated with Angiopep-PEG-PE/AmB achieved the greatest treatment ef ?cacy,which reached the highest AmB level in brain,reduced the brain fungal burden signi ?cantly,decreased histopathological severity and prolonged the median survival time.The increased treatment ef ?cacy could be attributed to the brain-targeting delivery system promoted AmB crossing the BBB and penetrating into the brain to reach the therapeutic concentration.The underlying mechanism was also explored in this work.Therefore,the brain-targeting delivery system could have potential and promising implications for treatment of intracerebral fungal infection.

1.Introduction

Cryptococcal meningitis or meningoencephalitis has a high morbidity and mortality in immunocompromised individuals,such as patients with AIDs [1,2].Amphotericin B (AmB),a hydrophobic antibiotic with broad antifungal spectrum,is commonly used in the treatment of cryptococcal meningitis and other invasive fungal infections of the CNS [3e 6].However,the blood e brain barrier (BBB)remains a pharmacologic barrier to AmB,regardless of formulations [7].The use of conventional deoxycholate formulation

of AmB is often limited by the need for intrathecal administration,since it is ineffective when given intravenously [8,9].Additionally,dose-related nephrotoxicity may severely restrict deoxycholate AmB utility.At present time,the commercial lipid-based formula-tions of AmB may offer new therapeutic alternatives due to reduced toxicities by altering the pharmacokinetics and tissue distribution of the drug [10].However,substantially higher dosages of lipid formulations of AmB did not exhibit signi ?cant improvement in clinical status,either in animal models [8,11]or clinical studies [12,13],which may be attributed to their poor penetration ef ?-ciency into the cerebrospinal ?uid (CSF)and brain tissue [7].Therefore,it is essential to develop a safe and effective carrier for AmB delivering into the CNS.

In our previous study,we have developed an effective brain-targeting drug delivery system,which was angiopep-2modi ?ed AmB incorporated PE-PEG based polymeric micelles (Angiopep-

*Corresponding author.Tel.:t862151980079.**Corresponding author.Tel.:t862152887966.

E-mail addresses:zhulp@https://www.360docs.net/doc/a615385300.html, (L.Zhu),jiangchen@https://www.360docs.net/doc/a615385300.html,

(C.

Jiang).

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Biomaterials

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https://www.360docs.net/doc/a615385300.html,/locate/biomaterials

Biomaterials 33(2012)6898e 6907

PEG-PE/AmB)[14].In aqueous media,the hydrophobic AmB molecules could be entrapped into the cores of micelles,which were able to signi?cantly increase the solubility of AmB.Angiopep-2,the ligand of low-density lipoprotein receptor-related protein (LRP)present on the BBB[15],was selected as targeted-molecule due to its high capacity of vectoring small molecules[16]or nanoparticles[17]into the brain tissue.The increased CNS pene-tration ef?ciency of Angiopep-PEG-PE/AmB polymeric micelles has been evaluated in normal animals,which was identi?ed primarily through transcytosis mediated by LRP.

P-glycoprotein(P-gp)is a type of ef?ux transporter widely expressed on the luminal surface of brain endothelial cells[18,19]. P-gp-mediated exclusion has been cited as a major factor hindering the ability of drugs to enter the brain,and thus an important source of therapeutic failure for potential CNS-active agents[20].Up to now,there has been no conclusive evidence to demonstrate that AmB is a substrate for P-gp.Interestingly,recent studies have shown that enhanced gastro-intestinal(GI)absorption of AmB through formulating it in the oral lipid-based delivery system, Peceolò,which could decrease P-gp-mediated drug ef?ux[21,22]. Another observation has shown that the expression level of P-gp in the duodenum increased by AmB inducing in Wistar rats after AmB treatment[23].These observations raised the question of whether the low ef?ciency of AmB penetration into the CNS was probably relevant to the P-gp exclusion.In other words,we hypothesized that the increased CNS penetration ef?ciency of AmB might partially be contributed to the overcome exclusion of P-gp by polymeric micellar delivery system.

2.Materials and methods

2.1.Materials

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000](PE-PEG)and1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000](PE-PEG-Mal)were purchased from Avanti Polar Lipids(Alabaster,AL,USA)and used without further puri?cation.Angiopep-2 (TFFYGGSRGKRNNFKTEEYC)was synthesized by Chinese Peptide Company(Hang-zhou,China).Rhodamine123ò(Rho123)was purchased from Sigma(St.Louis,MO, USA).40,60-diamidino-2-phenylindole(DAPI)and Wheat Germ Agglutinin Alexa Fluorò350Conjugation were purchased from Molecular Probes(Eugene,OR,USA). Amphotericin B(AmB)was gifted by Asia Pioneer,Shanghai.AMB,deoxycholate AmB(Asia Pioneer,Shanghai)and Amphotecò,liposomal formulation of AmB,the amphotericin B colloidal dispersion(ABCD)(Three Rivers Pharmaceuticals,New York City,USA)were provided by Huashan Hospital(Shanghai).Chemical agents were purchased from Sinopharm(Shanghai,China)as analytical grade preparations. Acetonitrile was purchased as HPLC grade from Dikma(Lake Forest,CA,USA).

2.2.Preparation of angiopep-2modi?ed(Angiopep-PEG-PE)or unmodi?ed(PEG-PE) polymeric micelles

The AmB-incorporated angiopep-2modi?ed(Angiopep-PEG-PE/AmB)or unmodi?ed polymeric micelles(PEG-PE/AmB)were prepared according to the previous optimized formulations as follows[14].Brie?y,a thin polymeric?lm was formed in a round-bottom?ask by removing the organic solvents from a solution of PE-PEG or PE-PEG mixed with PE-PEG-Maleimide(PE-PEG-Mal,40mol%of total polymers for modi?ed polymeric micelles)in chloroform and the optimized amount of AmB in methanol(1.7mg of AmB per10mg of polymers)by rotary evaporation. Fluorescently labeled polymeric micelles were obtained by rotary evaporation of chloroform solution of PE-PEG or PE-PEG/PE-PEG-Mal mixture containing0.5%w/w of the hydrophobic dye Rhodamine123ò.The dry polymeric?lm was dissolved in the10m M HEPES-buffered saline(HBS),pH7.4(10mg of polymers per1.5ml of buffer)and incubated at room temperature with stirring for2h to allow for complete equilibration.For angiopep-2conjugation,angiopep-2(PE-PEG-Mal: angiopep-22:1,mol/mol)was added to the solution and reacted for12h in darkness at room temperature.We used Ellman’s agent to measure the reactive ef?ciency,and the result suggested that nearly92%thiol groups could react with maleimide under this condition.

2.3.Cell lines

Brain capillary endothelial cells(BCECs)were kindly provided by Prof.J.N.Lou (the Clinical Medicine Research Institute of the Chinese e Japanese Friendship Hospital)[24].Primary BCECs were isolated from cerebral grey matter of CBA/J mouse brain by the research group of Prof.J.N.Lou[24].Primary BCECs were cultured as described previously[14,25].

Human neuroblastoma SH-SY5Y cells were grown in Dulbecco’s modi?ed Eagle medium containing100U/ml penicillin,100m g/ml streptomycin,and10%fetal bovine serum.Cells were incubated at37 C under a saturating humidity atmo-sphere containing5%CO2[26].

2.4.Animals

Male CD-1mice(the Sino-British SIPPR/BK Lab),six to eight weeks of age,were housed in ABSL-2Laboratory(Institute Pasteur of Shanghai Chinese Academy of Sciences),with?ve mice per cage,and provided irradiated food and acidi?ed water. Manipulations of animals were performed in accordance with the standards set by Pasteur Animal Ethics Committee.

2.5.Fluorescent and confocal microscopy study

2.5.1.The internalization by BCECs

BCECs were seeded at a density of104cells in cell culture dishes (35mm?10mm)(Corning-Coaster,Tokyo,Japan),incubated for24h,and checked under the microscope for morphology.For intracellular localization studies,BCECs were treated with?uorescent angiopep-2modi?ed or unmodi?ed polymeric micelles solutions containing5m M Rho123at37 C for30min[27,28],washed with PBS twice and incubated with1ml of Wheat Germ Agglutinin Alexa Fluorò350 conjugation(10m g/ml)10min at37 C for staining live cells membrane,according to manufacturer’s instructions.After washing,the?uorescence was immediately examined with confocal laser scanning microscope(Leica TCS SP5MP).

2.5.2.Brain uptake

Rho123-incorporated modi?ed polymeric micelles were injected into the tail vein of mice at a dose of100m g Rho123/mouse.One hour later,animals were anesthetized with i.p.10%chloral hydrate and were heart-perfused with20ml saline and4%paraformaldehyde solution respectively.The brains were removed,?xed in 4%paraformaldehyde for48h,and were placed in15%sucrose PBS solution for24h until subsidence,then in30%sucrose for48h until subsidence.Afterwards,excised brains were frozen atà80 C in OCT embedding medium(Sakura,Torrance,CA, USA).Frozen sections of20m m thickness were prepared with a cryotome Cryostat (Leica,CM1900,Wetzlar,Germany)and stained with300nM DAPI for10min at R.T. After washing twice with PBS,the sections were observed under confocal laser scanning microscope(Leica TCS SP5MP,Wetzlar,Germany).

2.6.Preparation of the inoculum for infection

Cryptococcus neoformans,a clinical strain,obtained from a patient with cryto-coccal meningitis and stored at Huashan Hospital(Shanghai),was used for all experiments.Before use,colony-forming units(CFU)in the stock were measured by cultures of serial dilutions on Sabouraud Dextrose Agar(SDA)plates at35 C for a minimum of3days.On the day of experiment,the inoculum was adjusted to the desired concentrations with sterile saline,and the CFU were con?rmed at the end of experiment,as described above.

2.7.Immunosuppressed murine model with intracerebral fungal infection

A murine model of CNS Cryptococcus infection was established as described previously with slight modi?cation[29e31].In brief,mice were given cyclophos-phamide i.p.at a dose of200mg/kg of body weight beginning at3days prior to inoculation and then every6days thereafter.Pancytopenia was documented by peripheral white blood cell counts beginning on the day of inoculation and throughout the experimental period[29].On the day of inoculation(Day0),mice were anesthetized with diethyl ether inhalation.A total105conidia in a volume of 20m l was inoculated intracerebrally at a point midline on the cranium,4e5mm posterior to the eyes.A27-gauge disposable needle was used to deliver the inoc-ulum to a depth of3e4mm.Mice were fully recovered within5min,and there were no deaths as a result of the inoculation procedure.

2.8.Therapy regimens

AMB,a colloidal suspension in deoxycholate and ABCD,the liposomal form of AmB were reconstituted according to the manufacturer’s instructions and diluted with sterile5%dextrose on the day of administration.Once-daily therapy was initiated on the day3after inoculation and given for3consecutive days(from day3 to day5).All mice were assigned randomly to six groups.Four groups of mice received AMB,ABCD,PEG-PE/AmB and Angiopep-PEG-PE/AmB polymeric micelle solutions at1mg AmB/kg bodyweight,one group of mice received ABCD at3mg AmB/kg bodyweight and one group of untreated mice served as controls.All drugs were administrated by slow intravenous push via the lateral tail vein.

K.Shao et al./Biomaterials33(2012)6898e69076899

https://www.360docs.net/doc/a615385300.html,parative analyses of brain penetration ef?ciency in murine model

To compare brain penetration ef?ciency of different formulations of AmB,we measured AmB concentrations in serum and brain tissues respectively.On day6 after3doses therapy as previously indicated in murine model,each group of5 animals were anesthetized and500m l blood samples were collected by retro-orbital puncture.After heart-infusion,the brains were removed and placed in preweighed Eppendorf tube and kept atà80 C until further processing.The blood samples were centrifuged at4000rpm for20min and the supernatant was collected and kept atà80 C until determination.The serum and brain samples were processed for the separation of AmB as described previously[14].The AmB concentrations in serum and brain tissues were determined by HPLC.

https://www.360docs.net/doc/a615385300.html,parative analyses of drug ef?cacy

Drug ef?cacy was determined by evaluating the following endpoints:measuring the survival rate and weight change,evaluating the brain fungal burden quantita-tively and histopathologic examination.For survival study,cages were inspected twice daily.All animals were observed for a total period postinfection of22days and bodyweight was recorded everyday.Each group included10mice.

For measurement of the brain fungal burden,on day6postinfection as previ-ously indicated,each group of10animals were enthanatized and heart-brain infused.The brains were removed aseptically and placed in sterile preweighed Eppendorf tube.After brain weights were determined,one volume of sterile saline was added and homogenized.Serial dilutions of the whole-brain homogenate were prepared for quantitative counts of CFU.The dilutions were plated as triplicates on SDA plates.The agar plate was incubated at35 C for48h and the numbers of CFU were recorded.The fungal burden in the brain is given as the log10CFU per gram of brain tissue.

For histopathologic examination,mice were enthanatized on day6and day9 postinfection and were heart-brain infused with sterile saline followed with4% buffered formalin.The brains and kidneys were removed and placed in4%neutral buffered formalin for histopathologic assessments.Periodic acid-Schiff(PAS)stains were performed on brain samples to highlight organisms and assess histopatho-logical severity,which was evaluated by a pathologist blinded to the treatment each animal had received.The paraf?n kidney sections were stained with Hematoxylin-Eosin(HE).

2.11.Cellular uptake,retention,and ef?ux study

SH-SY5Y or BCECs were seeded at a density of2?104cells/well in24-well plates (Corning-Coaster,Tokyo,Japan),incubated for48h,and checked under the micro-scope for con?uency and morphology.For cellular uptake assay,SH-SY5Y cells were rinsed twice with PBS pH7.4solution and then incubated with AMB,ABCD,PE-PEG/ AmB or Angiopep-PEG-PE/AmB polymeric micelles at AmB equivalent dose,2m g/ml, at37 C for30min.Cells were washed3times with cold PBS solution and further incubated with Triton X-100(1%,v/v)overnight at4 C for cellular lysis.The cell lysates were centrifuged at12,000?g for2min at4 C.The clear supernatant was collected for quanti?cation of AmB and cell protein.

The effect of P-gp on the uptake and retention behaviors of AmB was evaluated on BCECs.Brie?y,BCECs were preincubated with culture medium in presence or absence of15m M verapamil for2h[32].Then cells were incubated with AMB,ABCD or AmB-incorporated micellar formulations in presence or absence of15m M verapamil at37 C for1h.AmB was set to be2m g/ml.

For the ef?ux study,BCECs were incubated with2m g/ml AMB,ABCD or AmB-incorporated micellar formulations(AmB-equivalent dose)at37 C for1h and washed3times with PBS.Cells were further incubated with drug-free medium for another0,0.5,1or2h.At different time point after incubation,cells were washed3 times with cold PBS and lysed with300m l1%Triton X-100as mentioned above.The concentrations of AmB were evaluated by HPLC and standard solutions were prepared in the respective blank cell lysates,SH-SY5Y or BCECs.Protein concen-trations of the cell lysates were determined by the Bradford colorimetric assay with bovine serum albumin(BSA,Pierce)as a protein standard[33].The levels of AmB in cells were then normalized to protein amounts to ng AmB/mg cell protein.All experiments were performed in quadruplicate.

2.12.Processing HPLC analysis

HPLC analysis was performed with a modular liquid chromatograph system (Agilent TM).The mobile phase consisted of acetonitrile and10m M sodium acetate buffer,pH4.0(40:60,v/v)and the?ow rate was kept at1ml/min.The ef?uent was monitored at408nm[34].

2.1

3.Statistical analysis

The comparative survival rates were analyzed by a log rank test with GraphPad Prism software for Windows.A statistical analysis of CFU results was done by a Mann e Whitney U test.Statistical comparisons of AmB levels in brain or cells were done by Student’s tests.

3.Results

3.1.Fluorescent and confocal microscopy study

The internalization experiment in vitro was investigated in BCECs,which is shown in Fig.1.The cells treated with unmodi?ed polymeric micelles demonstrated very weak?uorescence signals (Fig.1A),while the signal of the ones treated with targeted poly-meric micelles was strong(Fig.1B).The Angiopep-PEG-PE poly-meric micelles internalization was con?rmed using a membrane ?uorescent marker(Wheat Germ Agglutinin Alexa Fluorò350 conjugation).The results clearly indicated that the major fraction of the Angiopep-PEG-PE polymeric micelles were cell-internalized and concentrated in cytosol(Fig.1B).

The confocal images allowed viewing of the green?uorescent signals distributed in the brain sections(Fig.2A).Free Rho123is not able to cross the BBB,because it is the substrate of P-gp expressed on the BBB[28].Green?uorescent spots,due to the Angiopep-PEG-PE polymeric micelles,concentrated in the perinuclear regions of brain cells(Fig.2B and C),which demonstrated that Angiopep-PEG-PE polymeric micelles could promote Rho123accumulating in the brain and further being internalized by brain

cells.

Fig.1.BCECs incubated with Rho123-incorporated unmodi?ed(A)and modi?ed(B)polymeric micelles(green)for30min,then with Wheat Germ Agglutinin Alexa Fluorò350 conjugation(blue),a membrane marker,for10min and observed by confocal microscopy.(For interpretation of the references to colour in this?gure legend,the reader is referred to the web version of this article.)

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3.2.Brain penetration ef ?ciency

Concentrations of AmB in serum and brain tissues respectively on day 6postinfection after the last of 3daily doses of AMB (1mg/kg/day),ABCD (1mg/kg/day or 3mg/kg/day),PEG-PE/AmB and Angiopep-PEG-PE/AmB polymeric micelles (1mg/kg/day each)are shown in Fig.3(A and B).

As Fig.3A shown,the serum concentration after administration of AMB was higher than after ABCD at 1mg/kg (*P <0.05).However,there was no statistically signi ?cant difference in serum levels between AmB polymeric micellar formulations and AMB or ABCD (1mg/kg or 3mg/kg).

The highest drug concentrations in brain parenchyma were achieved by Angiopep-PEG-PE/AmB,1.47-fold,1.6-fold and nearly 2.8-fold higher than unmodi ?ed polymeric micelles (*P <0.05),AMB (**P <0.01),and ABCD at 1mg/kg or 3mg/kg (***P <0.005)respectively (Fig.3B).No statistically signi ?cant differences in brain levels were observed among AMB and ABCD at 1mg/kg or 3mg/kg.

The ef ?ciency of AmB penetrating into the brain parenchyma from the blood circulation was evaluated by the brain/serum level ratio (Fig.3C).Angiopep-PEG-PE/AmB polymeric micelles-treated animals had the highest brain/serum ratio (**P <0.01vs.deoxy-cholate AmB,*P <0.05vs.PEG-PE/AmB and ABCD at 3mg/kg),whereas no signi ?cant differences among the other AmB formu-lations.Higher dose (3mg/kg)of ABCD did not exhibit the increased brain/serum ratio signi ?cantly.As evident by the analysis of the brain/serum ratio compared with the other formulations,Angiopep-PEG-PE/AmB polymeric micelles demonstrated an increased penetration ef ?ciency of AmB into the brain parenchyma.3.3.Burden of C.neoformans in the brain tissues

The recoveries of CFU of C .neoformans from brains of animals treated with various AmB formulations once-daily for three consecutive days are shown in Fig.4.As compared with the untreated group,only treatment with

Angiopep-PEG-PE/AmB

Fig.2.Confocal images of brain sections of mouse treated with Rho 123-incorporated Angiopep-PEG-PE polymeric micelles at 1h after intravenous injection (A).(B)and (C)display the enlarged view of the dashed regions in (A).Green spots represent modi ?ed polymeric micelles,while blue spots are cerebral nuclei.(For interpretation of the references to colour in this ?gure legend,the reader is referred to the web version of this

article.)

Fig.3.On day 6after inoculation,the levels of AmB in serum (A)and brain (B)were measured by HPLC.(C)shows the ratio of concentrations of AmB in brain to serum.Each group included 5animals.Data are expressed as the mean ?S.D.(*P <0.05,**P <0.01and ***P <0.001).

K.Shao et al./Biomaterials 33(2012)6898e 69076901

polymeric micelles at 1mg/kg and ABCD at 3mg/kg led to a statis-tically signi ?cant quantitative reduction in the residual fungus burden in brain tissues.There were no signi ?cant differences comparing AMB and ABCD treatment,nor between the two doses of ABCD.Those treated with Angiopep-PEG-PE/AmB polymeric micelles at 1mg/kg had the lowest mean burden,with P values compared with other groups as follows:untreated group,P ?0.0031;PEG-PE/AmB at 1mg/kg,P ?0.0232;AMB at 1mg/kg,P ?0.0021;ABCD at 1mg/kg,P ?0.0115;ABCD at 3mg/kg,P ?0.0433.3.4.Histopathologic examination

Histopathological assessment of brain was performed with samples of all animals on day 6postinfection (Fig.5).However,it was failure to collect the brain sample of untreated group due to it produced 90%mortality on day 9postinfection (Fig.6).Histo-pathological study revealed a large amount of coccidioidal conidia scattered in the subarachnoid space.The presence in the brain parenchyma of macro-and micro-abscesses with necrosis (Fig.5G,red arrow in the web version)and diffused invasion of conidia were observed (Fig.5G,black arrow).Comparison the histopathological severity of AMB treated group on day 6and on day 9postinfection,the parenchymal area had a more diffuse pattern of in ?ltration of the fungal conidia (Fig.6F,black arrow)that was associated with acute necrosis,demonstrating that the progressive process of the disease even under the condition of drug administration.In contrast,the Angiopep-PEG-PE/AmB treated group showed the reduced amount of conidia in subarachnoid space and decreased parenchymal invasion,both on day 6and on day 9postinfection (Fig.5F and L,Fig.6E and J),meaning that Angiopep-PEG-PE/AmB polymeric micelles was increasingly penetrated into the CNS after systemic circulation and effectively reduced the histological severity of the disease.

3.5.Nephrotoxicity examination

Staining of paraf ?n kidney sections of the three repeated-dose of AMB at 1mg/kg revealed nuclear shrinkage of renal tubular

epithelial cells,and some nuclei appeared fragmented and dis-solved (Fig.7A).ABCD-treated mice showed moderate congestion in the medulla (Fig.7B and C),whereas mice treated with AmB polymeric micellar formulations showed mild conges-tion (Fig.7D and E),which suggested that the polymeric micellar formulations may reduce the nephrotoxicity compared with deoxycholate AmB.

3.6.Survival and weight change

The survival curves of the various groups are presented in Fig.8.The established model proved to be lethal to untreated group,with 100%mice succumbing to infection between days 6and 10post-infection (Fig.8A).Statistical comparison of survival by time was done by the log rank test.The results suggested that all treatment regimens had signi ?cant ef ?cacy in the prolongation of survival compared with untreated group (***P <0.001).The group received with Angiopep-PEG-PE/AmB achieved the most effective treatment ef ?cacy (***P <0.001vs.PEG-PE/AmB or AMB at 1mg/kg,**P <0.01vs.ABCD at 1mg/kg,*P <0.05vs.ABCD at 3mg/kg),in which three of ten animals survived to the termination of the experiment,day 22.However,no signi ?cant differences among the other groups were observed (P >0.05)and ABCD at 1mg/kg and 3mg/kg doses showed equivalent ef ?cacies (P >0.05),which was in consistent with the observation of brain fungal burden.

Untreated animals lost weight rapidly,losing more than 20%of their initial weight within eight days.Body weights of Angiopep-PEG-PE/AmB treated group showed an initial rise after three days treatment followed by a progressively drop beginning on day 8.However,all other treated groups showed a continued drop in weight,even treated with ABCD at 3mg/kg (Fig.8B).3.7.Effect of verapamil on the uptake by BCECs

Effect of P-gp inhibitor verapamil on the uptake of AmB by BCECs was shown in Fig.9.Blockade of P-gp activity by verapamil,the internalization of AMB by BCECs was signi ?cantly increased (***P <0.001).In contrast,the AmB micellar formulations and AmB lipid-based formulation were insusceptible to verapamil inhibition (P >0.05).

3.8.Cellular retention of AmB-incorporated micellar formulations BCECs were incubated with AMB,ABCD or AmB-incorporated micellar formulations for 1h and then reincubated with fresh drug-free cell culture medium for additional 0.5,1,and 2h,and then analyzed by HPLC to measure the drug retention in the cells.As Fig.10indicated,in the group treated with AMB,only 30%AmB was retained at 2h.In contrast,upon addition of AmB polymeric micellar formulations at an equivalent dose of AMB,the retention of AmB in BCECs were signi ?cantly increased,remaining at a high intracellular level of 60%after 2h incubation,similar with the AmB lipid formulation.3.9.Uptake by SH-SY5Y

We studied the uptake characteristics of AmB-incorporated polymeric micelles by neurons,SH-SY5Y.As shown in Fig.11,compared with ABCD,the cellular uptake of Angiopep-PE-PEG/AmB micelles was remarkably increased 2.5fold (***P <0.001).As expected,the uptake of Angiopep-PEG-PE/AmB was prior to that of PEG-PE/AmB,nearly 1.5-fold higher (**P <0.01).This is consis-tent with our previous observation in BCECs incubated for 30min [14],suggesting that Angiopep-PEG-PE/AmB polymeric micelles may also undergo LRP-mediated internalization in

neurons.

Fig.4.Scattergrams of CFU recovered from the brains of surviving mice on day 6postinfection.Each data point corresponds to the number of log 10CFU/g brain for an individual mouse.The bars represent median group values.In the group consisting of untreated mice euthanatized on day 6,there were 7animals,and all other groups included 10animals (*P <0.05and **P <0.01).

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4.Discussion

We have successfully developed an effective brain-targeting polymeric micellar system with angiopep-2modi ?ed to improve the CNS permeability of AmB through the LRP-mediated trans-cytosis [14].In the current work,we show that angiopep-2modi-?ed polymeric micelles can rapidly and ef ?ciently delivery AmB into the brain tissue so as to strikingly enhance the treatment ef ?cacy against intracerebral fungal infection compared with unmodi ?ed polymeric micelles and AmB commercial formulations.

We reliably established the immunosuppressive murine model with intracerebral fungal infection for assessing the CNS penetra-tion ef ?ciency and investigating antifungal ef ?cacy [7,30,35].The resulting model was evidenced by large numbers of con ?uent foci in subarachnoid space and large coccidioidal spherules in the brain parenchyma of untreated mice on day 6after inoculation (Fig.5A and G),which was consistent with previous studies [29,31].Treatment of parenchymal fungal infection of the CNS may re ?ect potency and ef ?ciency of drug delivered into the brain tissue [36].

The determination of AmB levels in brain or serum after three consecutive days of treatment was performed to evaluate the CNS penetration ef ?ciency of different AmB formulations.The median AmB serum concentrations of modi ?ed or unmodi ?ed polymeric micelles were similar to that of AMB and ABCD (not signi ?cantly different).At an equal dose level (1mg/kg),AMB showed about 2-fold higher serum concentration than ABCD.Despite of a 3-fold increase in the dose of ABCD,it did not produce a proportional increase in serum concentration of AmB (Fig.3A),consisted with previous study in rats after repeated dosing [37].The AmB level in brain (Fig.3B)was determined after processing the heart-brain infusion with a drug-free solution,excluding the drug content in the vessels in the parenchyma which could have contributed to

the

Fig.5.Photomicrographs (?200)of subarachnoid space (A e F)and brain parenchyma (G e L)showing the effect of treatment ef ?cacy for intracranial C.neoformans infection.On day 6after inoculation,a histopathological analysis of periodic acid-schiff-stained (PAS)brain sections were performed.Groups:untreated (A and G),AMB 1mg/kg (B and H),ABCD 1mg/kg (C and I)ABCD 3mg/kg (D and J),PEG-PE/AmB 1mg/kg (E and K)and Angiopep-PEG-PE/AmB (F and L).

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Fig.6.Photomicrographs (?200)of subarachnoid space (A e E)and brain parenchyma (F e J)showing the effect of treatment ef ?cacy for intracranial C.neoformans infection.On day 9after inoculation,a histopathological analysis of periodic acid-schiff-stained (PAS)brain sections were performed.Groups:AMB 1mg/kg (A and F),ABCD 1mg/kg (B and G)ABCD 3mg/kg (C and H),PEG-PE/AmB 1mg/kg (D and I)and Angiopep-PEG-PE/AmB (E and

J).

Fig.7.Nephrotoxicity assessment of immunosuppressed mice intracranially infected with 105cfu/animal and treated with AMB 1mg/kg (A),ABCD 1mg/kg (B),ABCD 3mg/kg (C),PE-PEG/AmB 1mg/kg (D)or Angiopep-PEG-PE/AmB 1mg/kg (E)from day 3to day 5after infection.The paraf ?n sections were stained with Hematoxylin-Eosin (HE)(?400).

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values they attributed to the brain [11].Therefore,the brain level of AmB in this study to some extent re ?ected the AmB concentration in brain parenchyma.The group received Angiopep-PEG-PE/AmB polymeric micelles achieved the highest AmB level in brain tissue among all the treated groups,although they had similar serum concentrations (Fig.3B and C).Our data suggested the intriguing possibility that angiopep-2modi ?ed polymeric micelles might be able to delivery AmB into the CNS and to promote accumulation in the brain parenchyma from the blood circulation.

For treatment ef ?cacy evaluation,our results showed that all regimens reduced the numbers of CFU in brain tissue and pro-longed survival days compared with untreated animals (Figs.4and 8A).Most encouraging results were demonstrating that intrave-nously administrated Angiopep-PEG-PE/AmB polymeric micelles at 1mg/kg showed signi ?cantly improved clinical parameters,with substantially cleared burdens of C.neoformans in the brain tissue,reduced histopathological severity,prolongation of survival and retarded the weight loss.These parameters were even better than those of group treated with ABCD at 3mg/kg.Interestingly to note that,comparison of data from groups treated with ABCD at 1mg/kg and 3mg/kg demonstrated that increased dose did not result in improved antifungal ef ?cacy (no signi ?cant difference).Similar conclusions regarding the lack of additional bene ?t of higher dosages of liposomal form of AmB were obtained in previous studies [8,12].This phenomenon may be contributed to the poor CNS permeability of liposomal form of AmB,as observed in the AmB brain/serum ratio mentioned above (Fig.3C).Thus,we may ?gure out a strong correlation of clearance of C.neoformans from brain tissue and prolongation of survival with the ef ?ciency of

AmB

Fig.8.Survival curves (A)and weight changes (B)of immunosuppressed mice intracranially infected with 105cfu/aminal and treated with AMB,ABCD and AmB-incorporated micellar formulations at 1mg/kg or with ABCD at 3mg/kg from day 3to day 5after

infection.

Fig.9.Effect of P-gp inhibitor verapamil on the uptake of AmB by BCECs.Cells were preincubated with culture medium in presence or absence of 15m M verapamil for 2h.Cells were incubated with AMB,ABCD and AmB-incorporated polymeric micellar formulations in presence or absence of 15m M verapamil at 37 C for 1h.AmB was set to be 2m g/ml.Data are expressed as the mean ?S.D.(n ?4)***P <0.001.

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delivered into the CNS [38].The most excellent ef ?cacy obtained by Angiopep-PEG-PE/AmB polymeric micelles indicated that the brain-targeting delivery system could improve AmB permeability into the CNS and achieve the therapeutically effective drug levels in brain tissue,even at the dose of 1mg/kg.

It will be of great interest to decipher the possible mechanism of the increased CNS penetration ef ?ciency of Angiopep-PEG-PE/AmB polymeric micelles.The majority aspect may be attributed to the high ef ?cient transcytosis capacity and parenchymal accumulation through LRP-mediated pathway [15],proved by angiopep-2effec-tively facilitate polymeric micelles being internalized by BCECs [16]and neurons (Fig.11).Meanwhile,we hypothesized that low CNS penetration ef ?ciency of AmB molecule may be relevant to the P-gp ef ?ux as well [21].Our results showed that ABCD and AmB poly-meric micellar formulations displayed insusceptible to blockade of P-gp activity by verapamil (Fig.9).In addition,ABCD and AmB polymeric micellar formulations strikingly increased the retention of AmB in cells (Fig.10),which all probably suggested that P-gp fail to export AmB when it is encapsulated in the nanoparticles [32,39,40].5.Conclusion

In summary,the angiopep-2modi ?ed brain-targeting poly-meric micelles could effectively delivery AmB into the CNS and maintaining higher drug levels in brain parenchyma.Considering the dose-dependent nephrotoxicity of AmB,the brain-targeting polymeric micelles may be the optimal alternative to achieve equivalent or greater ef ?cacy even at lower dosage.Therefore,the brain-targeting delivery system could have potential and promising implications for treatment of intracerebral fungal infection.Acknowledgments

The authors are grateful to Prof.Qibin Leng and Dr.Chunfu Yang for helpful advice in this work.We acknowledge Institute Pasteur of Shanghai Chinese Academy of Sciences for providing access to the BSL-2Laboratory.This work was supported by National Nature Science Foundation of China (30973652),Program for New Century Excellent Talents in University and National Nature Science Foun-dation of China (81071333).References

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