Nanoparticle structure transformation of m PEG grafted chitosan with rigid backbone induced by a-cyclodextrin

Lei Huang,Jiaojiao Chen,Muye He,Xinyu Hou,Yien Lu,Kaiyan Lou,c,Feng Gaoa,,c,*

a Shanghai Key Laboratory of Functional Materials Chemistry,EastChina University of Science and Technology,Shanghai 200237,China

b Department of Pharmaceutics,School of Pharmacy,East China University of Science and Technology,Shanghai 200237,China

c Shanghai Key Laboratory of New Drug Design,EastChina University of Science and Technology,Shanghai 200237,China

Key words:a-Cyclodextrin m PEG Grafted chitosan Self-assembly Nanoparticle Drug delivery

ABSTRACT Thispaper presented an interesting nanoparticle-based drug delivery system with morphology transition behavior depending on the content of exposed PEG chain on the particle surface,which is adjustable by addition of different amount of cyclodextrin(a-CD).The effect of a-CD inclusion to the self-assembly behavior of methoxy polyethylene glycol(m PEG)grafted chitosan(CS)was studied.The results showed that the m PEGgrafted chitosan(m PEG- g-CS)forms self-assembled nanoparticles with either micelle or hollow sphere morphology depending on the ratio of a-CD to m PEG,as characterized by atomic force microscopy(AFM),transmission electron microscopy(TEM),and X-ray diffraction(XRD).Their sizes and zeta potential increased from 257.6 nmto 768.2 nm and from+4.5 m V to+11.6 m V,respectively,with the increasing amount of a-CD.The correlation between zeta potential and particle size of a-CD/m PEG- g-CS nanoparticles indicated varied PEGdensity on surface of nanoparticles.Based on the above experimental observations,a likely mechanism for the morphological transition of the rod-coil graft copolymer m PEG- g-CSwas proposed.

Recently,morphology-controlled self-assembly of block and graft copolymers have attracted much interest,due to their potential applications in many fields such as in biomaterials and drug delivery[1–3].Block or graft copolymers can be selfassembled to form micelles in their selective solvents.Compared with linear block copolymers,graft copolymer can add considerable functionalities on the branched chains with great advantage of convenient chemical modifications in advance of the self-assembly process[4,5].However,in most cases,amphiphilic graft copolymers tend to form compound spherical micelles[6,7],w hereas amphiphilic linear block copolymers can organize into abundant morphologies such as spheres,rods,vesicles,[13_TD DIFF]etc.[8–10].Among many self-assembled morphologies,hollow spheres have draw n great attentions recently due to their unique large encapsulation capabilities with potential applications in the controlled release and targeted drug delivery[11–13].Polymer-based hollow spheres of various dimensions can be fabricated by different methods,such as emulsion polymerization[14],layer by layer method[15],selfassembly of polyelectrolytes or rigid-coil copolymer[16,17].Among various methods,self-assembly of rigid-coil copolymer presents the most popular one due to its simple preparation procedure[18],as hollow spheres could be obtained directly in their selective solvent through self-assembly[19].

Chitosan(CS),an amino-polysaccharide from deacetylation of chitin,has been investigated for biomedical applications due to its low-toxicity,good biocompatibility and biodegradability[20–23].Self-assembly behavior of CS-based biomaterials and their applications for drug delivery have been the focus of our research[24–34].One major draw back of chitosan is its low solubility in physiological condition(p H 7.4),which greatly limits its utilities as it is only soluble in a few dilute acidic solutions such as in acetic acid and in dilute hydrochloric acid[20].To improve chitosan solubility,methoxy polyethylene glycol(m PEG),a well-adopted hydrophilic,biocompatible,and biodegradable polymer for stability enhancement of nanoparticles,sustained release of protein,and reduction of severe side effects such as cytotoxicity in physicochemical environment[35–38],could be grafted onto chitosan polysaccharide backbone.Moreover,the rigidity of the PEG chain could be further adjusted by incorporation of a-cyclodextrin(a-CD).First reported by Harada[39],PEG and a-CD forms rigid rod-like structure by threading a-CD molecules onto PEG chain.However,the further self-assembly of the formed a-CD/PEG rigid rod-like structure to more complex morphologies were seldom studied in literature[35,41].Recently,Zhang and coworkers[40]managed to use chitosan as the coil section and a-CD/PEGcomplex as the rigid section to afford hollow spheres loaded with magneto fluid in aqueous solution.We envisioned that the amount of a-CDmay be crucial for the formation of hollow spheres,but has not yet been fully explored.

Herein,we fully investigated the effect of a-CD inclusion to the self-assembly behavior of m PEGgrafted chitosan.The a-CD/m PEG- g-CSsystem forms different morphology of nanoparticle and PEG density on the surface of nanoparticle at different ratio of[CD]/[EG](Scheme 1).As far as we know,the current work presents the fi rst report on the change of aggregation behavior of m PEG- g-CSgraft copolymer induced by the different concentration of a-CD.

In the present studies,m PEG- g-CS was synthesized and characterized(Fig.S1 in Supporting information).The1H NMR spectra of chitosan and m PEG- g-CS were show n in Fig.S2(Supporting information).The proton assignment of chitosan(Fig.S2A)was as follow s(ppm):2.93(CH,carbon b of glucosamine ring),3.4–3.8(CH,carbon c,d,e and f of glucosamine ring).Compared with chitosan,the characteristic proton signals of m PEG- g-CS(Fig.S2B)appeared in the range of 3.1–3.5 ppm.The peak at around 2.97 ppm was the proton signal of methoxyl of m PEG- g-CS.Therefore,degree of substitute(DS)of m PEG moiety could be calculated by comparing the ratio of methoxyl of m PEG protons(2.97 ppm)to sugar protons(2.72 ppm,CH,carbon 2 of glucosamine ring).The DS could be calculated by follow s:The calculalted DSof m PEG moiety on m PEG- g-CSwas 21.5%.Fig.S3(Supporting information)showed the Fourier transform infrared spectroscopy(FT-IR)spectra of chitosan(a)and m PEG- g-CS(b).The chitosan spectrum(Fig.S3A)showed characteristic bands of amide I(1683 cm?1)and amide II(1587 cm?1).The peaks at 3420 cm?1in chitosan scaffolds corresponded to O??H stretch overlapped with N-H stretch.For m PEG- g-CS(Fig.S3B),bands of amide I(1683 cm?1)and amide II(1587 cm?1)observably decreased.Furthermore,the increased intensity of the peaks at around 2920,1739 and 1100 cm?1indicated the CH2,C?O and C??O??C stretch of m PEG.These results showed that the amino groups of chitosan were successfully substituted by m PEG groups.

p H-dependent solubility study of m PEG- g-CS(Fig.1A)revealed that solubility of the prepared polymer m PEG- g-CSat various p H from 5 to 11 in aqueous solution was studied and compared with chitosan.At p H 5,both chitosan and m PEG- g-CSshowed an optical transmittance over 98%,indicating good solubility for both polymers.When p H>6.1,chitosan started to form a milky solution due to reduced solubility and formation of large aggregates.In contrast,m PEG- g-CS remained a clear solution with an optical transmittance more than 90%,indicating a good solubility of m PEG- g-CS at the p H range tested from 5 to 11.Clearly,the introduction of m PEG to the main chain of chitosan greatly enhances the polymer’s solubility at p H over 6.1 including physiological p H at 7.4.

Then,hollow spheres were prepared by adding a-CD solution into m PEG- g-CS,the threading of a-CD onto the fl anking m PEG chains of the m PEG- g-CS copolymer,and inclusion formation between the a-CD and m PEG- g-CSwere further con fi rmed with X-ray pow der diffraction studies.As show n in Fig.S4(Supporting information),the pattern of a-CD/m PEG- g-CS particles were similar to complex of a-CD/m PEG inclusion(2u=20.0?)[39],which has been reported to have a rod channel structure,while the m PEG- g-CS crystalline peaks(2u=19.1?and 23.2?)and a-CD crystalline peak(2u=21.7?)were absent.This implied that a-CD rings are stacked along the graft PEGchain axis to form a channeltype crystalline structure.As a result,the a-CD/m PEG- g-CS has both rod block(a-CD/m PEG complex)and coil block(protonated chitosan chain).It was reported that polymer with rod-coil structure prefers parallel packing to achieve the most efficient space-fi lling and resulted in the formation of hollow spheres[19].

To explore the effect of molar ratio of a-CD to m PEG- g-CS to properties of a-CD/m PEG- g-CS nanoparticles,a series of a-CD/m PEG- g-CS nanoparticles with different molar(0,0.1,0.25,0.5,0.75,1.0)ratio of[CD]to[EG],[CD]indicated molar concentration of a-CD and[EG]indicated molar concentration of ethylene glycol unit,were prepared in aqueous solution(p H 6.0).As show n in Fig.1B,particle size of a-CD/m PEG- g-CS nanoparticles increased from 257.6 nm to 699.7 nm as the molar ratio of[CD]/[EG]increased from 0 to 1.0.To explain this phenomenon,a possible mechanism was proposed as show n in Scheme 1.m PEG- g-CSwas able to form micelle composed of an inner core mainly consisting of chitosan with entangled intramolecular hydrogen bonding network within and an outside corona consisting of mainly m PEG chains for hydrophilic interaction with solvent molecules w hen no a-CD was added[30].Upon addition of a-CD,the complexation of a-CD molecules with m PEG chains through sliding into the PEG chains forms rigid rod-like complexes[39],which tend to stick to each other and exclude solvent molecules,resulting in translocation of m PEG chains for better space-fi lling,reduced shielding of positive chargesof chitosan,and the grow th of a cavity within each nanospheres[19].

As show n in Fig.1B,no signi fi cant physicochemical property change occurred until the molar ratio of[CD]/[EG]reached 0.25.

Schem e 1.Schematic illustration of the formation of a-CD/m PEG- g-CSnanoparticles.

Fig.1.(A)The influence of p H on w ater solubility of chitosan and m PEG- g-CS.(B)The relationship of particle size and zeta potential of a-CD/m PEG- g-CSnanoparticle with different molar ratio of a-CD to m PEG- g-CSin p H 6.0 aqueous solution.(C)Correlation between zeta potential and particle size of a-CD/m PEG- g-CSnanoparticle with molar ratio of a-CD to m PEG- g-CSfrom 0 to 1.0 in p H 6.0 aqueous solution.(D)The size change of m PEG- g-CSmicelles and a-CD/m PEG-g-CS([CD]/[EG]=0.25)nanoparticles in p H 7.4 aqueous solution at 4?C.Data were expressed as mean?SD(n=3).

And then starting from[CD]/[EG]=0.25,a-CD/m PEG-g-CS nanoparticles([CD]/[EG]=0.25,0.5,0.75,1.0)exhibited increased zeta potential from+4.5 m V to+11.6 m V along with the increased size from 495.2 nm to 669.7 nm.The increased zeta potential showed reduced shielding of the positive charges of chitosan,suggesting the reduced density of uncomplexed m PEGchain on the surface of nanoparticles.Meanwhile,the increased particle size indicated the grow th of hollow sphere in the nanoparticles due to increased a-CD/m PEGcomplexation.Fig.1Cshowed the correlation between zeta potential and particle size of a-CD/m PEG- g-CSnanoparticles,indicating varied PEG density on surface of nanoparticles and m PEG chain can transferred from outside to inside,leading to exposed positive charge of chitosan and increased particle size by efficient space-fi lling packing of a-CD/m PEG complexes.

To investigate the effect of p H on the self-asssembly behavior of a-CD/m PEG- g-CS nanoparticles,nanoparticles were prepared under various p H(4.0,5.0,6.0,6.8,7.4,8.0).As show n in Table 1,a-CD/m PEG-g-CS([CD]/[EG]=0.25)nanoparticles,w hen p H value increased from 4.0 to 8.0,the particle size and the zeta potential gradually decreased from 666.1 nm to 308.5 nm and from+28.3 m V to+2.0 m V,respectively.This phenomenon could be rationalzied as the follow ing.a-CD/m PEG- g-CS([CD]/[EG]=0.25)nanoparticles contain a highly compact protonated inner chitosan core surrounded by a relative hydrophilic m PEG corona.At lower p H,particle size of a-CD/m PEG- g-CS([CD]/[EG]=0.25)nanoparticles were increased likely due to the increased electronic repulsion of protonated amino groups within the chitosan core.As for a-CD/m PEG-g-CS ([CD]/[EG]=1.0) nanoparticles,the particle size increased gradually from around 504.8 nm to 768.2 nm,while the zeta potential decreased gradually from+31.1 m V to?3.4 m V w hen the p H value increased from 4.0 to 8.0.a-CD/m PEG-g-CS([CD]/[EG]=1.0)nanoparticles contained a hollow cavity inside due to efficient space-fi lling packing of rod-shaped a-CD/m PEG- g-CS,causing few free PEG chains and chitosan chains stretchedoutside as a stablizing corona.The chitosan(pK a=6.5)chains were poor soluble because of the deprotonation of amino groups.Therefore,at high p H region(p H>6.5),the a-CD/m PEG- g-CS nanoparticles were not stable and macrocosmic precipitation took place.As p H value decreased,the amino group was ionized and then the a-CD/m PEG-g-CS([CD]/[EG]=1.0)nanoparticles became more and more stable.

Table 1 Physicochemical properties of a-CD/m PEG- g-CS nanoparticles.Data were expressed as mean?SD(n=3).

The morphology of m PEG- g-CS micelles were studied by transmission electron microscopy (TEM) [23_TD DIFF]and atomic force microscopy(AFM).The results were show n in[24_TD DIFF]Figs.2A,C,and E.Micelles had spherical morphology and maintained in nonaggregated state.It should be noticed that the size of m PEG- g-CS estimated from TEM is about 50–100 nm,which was much smaller than that from dynamic light scattering(DLS).The discrepancy of nanoparticle size measured between DLS and TEM was likely due to the shrinkage of nanoparticles under vacuum condition since TEM was performed under high vacuum.However,AFM images showed the diameters of m PEG- g-CS nanoparticles are around 300–350 nm,which was even larger than that from DLS(257.6 nm).The seemingly discrepancy might be caused by the different sample preparation and measurement condition since DLSwas performed in aqueous solution,while the AFM image were taken in dried sample in which nanoparticles tended to be fl atten[25_TD DIFF]42].Upon addition of a-CD into m PEG- g-CS solution,hollow spheres could be obtained as show n in the TEM image(Fig.2B).The contrast between central cavity and outer layer clearly indicated the presence of hollow sphere structure of the nanoparticles[17].From AFM studies([24_TD DIFF]Figs.2D and F),the height/particle size ratio of a-CD/m PEG- g-CS(around 1:2)nanoparticle([CD]/[EG]=0.25)washigher than that of m PEG- g-CS(around 1:8),re fl ecting increased rigidity due to threading of a-CD into m PEG chains of m PEG- g-CSnanoparticles.

Stability study(Fig.1D)showed that m PEG- g-CSnanoparticles showed good stability in the fi rst seven days without much increase of particle size,while the particle size significantly increased from the day 7 to the day 10.In contrast,a-CD/m PEG-g-CS([CD]/[EG]=0.25)nanoparticles showed good stability up to ten days.The increased stability could be rationalized by the enhanced rigidity of nanoparticles upon addition of a-CD.Apparently,the conformational fl exibility of m PEG- g-CS makes it easier to dissociate than a more rigid a-CD/m PEG- g-CS.

In conclusion,we presented an interesting nanoparticle-based drug-delivery system,a-CD/m PEG- g-CS,with morphology transition behavior depending on the different amount of a-CD added.The effect of a-CD inclusion on the self-assembly behavior of m PEG grafted chitosan was studied.The results showed that the m PEG- g-CS forms self-assembled nanoparticles with either micelle or hollow sphere morphology depending on the ratio of a-CD to EG,as characterized by DLS,AFM,TEM,and X-ray diffraction(XRD).The increase of nanoparticle size and zeta potential upon addition of a-CD correlated well with the decreasing amount of PEGdensity on the surface of nanoparticles.Due to its unique p H dependent properties and large internal cavity,the a-CD/m PEG- g-CShollow spheres showed promising as a novel nanocarrier system for delivery of protein drugs.The method of controlling the PEG density on surface of nanoparticle by a-CD paved a path to investigate the in vivo circulation time of nanoparticle in the future.

Fig.2.The TEM and AFM image of m PEG- g-CSmicelles(A,C,E),a-CD/m PEG-g-CS([CD]/[EG]=0.25)nanoparticles(B,D,F).

Acknow ledgments

This work was supported by Science and Technology Commission of Shanghai Municipality(Nos.17ZR1406600,10DZ2220500,11DZ2260600)and National Natural Science Foundation of China(No.21577037).

App endix A.Supp lem entary data

Supplementary data associated with this article can be found,in the online version,at https://doi.org/10.1016/j.cclet.2017.12.012.