Biomineralization-Based Virus Shell-Engineering: Towards Neutralization Escape and Tropism Expansion

www.advhealthmat.dewww.MaterialsViews.com

CO

MM

UN

ICATIO

Xiaoyu Wang,* Yongqiang Deng, Shihua Li, Guangchuan Wang, Ede Qin, Xurong Xu, Ruikang Tang,* and Chengfeng Qin*

Biomineralization-Based Virus Shell-Engineering: Towards Neutralization Escape and Tropism Expansion

N

In recent years, viruses have become fascinating materials due to nanoscale size, symmetric structure, monodispersive char-acter and delicate properties. [ 1 , 2 ] The major drawbacks in the development of virus-based toolkits for the biomedical appli-cations are the restricted tissue tropism and inactivation by the pre-existing immunity. [ 3 ] Viral surface engineering is an ideal approach to tailor the virus with desired functions and meanwhile preserve the natural properties of virus. However, most of the current viral engineering methods, such as genetic recombination, PEGylation and covalent modulations are irre-versible, which potentially interfere with the viral production, infection and transduction processes, [ 4–6 ] further resulting in the insuffi cient epitope-shielding effect and the undesired inactivation. [ 7 , 8 ] It is anticipated that the non-covalent method, comparing with the covalent and genetic strategies, is more simple, reversible, and environmental friendly for the viral reconstruction without disturbing its biological properties. Previously, we have adopted layer-by-layer techniques for the fabrication of virus-polyelectrolyte core-shell structure, but the treated virus fail to release from the nondegradable shell during cellular uptake. [ 9 ] Although some of the polyelectrolytes are biodegradable, they are not optimal materials for viral engi-neering, because their encapsulation of virus can lead to poor loading effi ciency, slow release, and subsequently low transfec-tion effi ciency. [ 10 ] The polymer modifi cations still play limited roles in non-covalent viral engineering. Therefore, it is tricky to manipulate the interplay of inner virus and the outer shells to achieve the bio-reversible modifi cations, probably due to the non-degradable and distinct features of the outer layers. A more elegant non-covalent based surface engineering strategy is urgently required to solve these issues and guarantee the biomedical applications.

© 2012 WILEY-VCH Verlag Gm

DOI: 10.1002/adhm.201200034

Dr. X. Wang , G. Wang , Dr. X. Xu , Prof. R. Tang Department of ChemistryZhejiang UniversityHangzhou, Zhejiang, 310027 (China) E-mail: [email protected]; [email protected] Dr. Y. Deng , S. Li , Prof. E. Qin , Prof. C. Qin State Key Laboratory of Pathogen and BiosecurityBeijing Institute of Microbiology and EpidemiologyBeijing, 100071, ChinaE-mail: [email protected] Dr. X. Wang Qiushi Academy for Advanced StudiesZhejiang UniversityHangzhou, Zhejiang, 310027, China

Adv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201200034

Biomineralization processes in organisms inspire the use of technique to fabricate smart inorganic-based hybrid bio-materials. [ 11 ] Biomimetic mineralization becomes an effective physicochemical tool to engineer the bio-interface of living organisms, such as cells and embryo, by using non-living mate-rials, blurring the boundary between living and non-living sys-tems. [ 12 , 13 ] Calcium phosphate (CaPi), analogous to human bone and teeth, is one of the most important biominerals because of excellent biocompatibility, non-toxicity, biodegradability, which is widely used for various biological applications such as trans-fection agent, adjuvant and drug delivery. [ 14–16 ] Up to now, CaPi has been used for the non-covalent deposition on the cellular matrix with complete coverage to produce shell-like structures; it is also reported that CaPi artifi cial shell is protective in hos-tile solutions and degradable under acid condition in vitro. [ 17 ] This encourages us to utilize CaPi to engineer the virus biosur-face and further investigate in vitro and in vivo protection and deprotection processs, which have not been reported so far.

Combining the above two aspects, we develop, in this com-munication, a novel concept of biomineralization based virus shell-engineering (BVSE) to modify virus with a CaPi shell by in situ biomineralization. It is expected that the strategy pro-vides the virus-CaPi hybrid with the distinct physical, chemical and biological characteristics of the native virus. Specifi cally, we applied adenovirus serotype 5 (Ad5) as a model virus, since it is a world-wide prevalent human virus and the most poten-tial viral vector for gene therapy and vaccination in clinic. [ 18 , 19 ] The application of Ad5 as vector system encountered several obstacles. First, it is highly cellular primary Coxsackievirus and adenovirus receptor (CAR) dependent, which is invalid for the CAR-defi cient cells. [ 20 ] Second, Ad5 exhibited the specifi c liver tropism and hampered the delivery of such virus to other thera-peutic tissues. [ 21 , 22 ] Third, the high prevalence of pre-existing immunity to Ad5 in human populations, particularly in the developing world, may substantially limit the immunogenicity and clinical utility of recombinant Ad5 as the vector-based vac-cines. [ 23 ] Hence, in the present study, we are interested to know whether or not the BVSE could circumvent the above problems and reveal a therapeutic potential.

Due to the abundant anionic peptides on virus surface, the zeta potential of recombinant Ad5 encoding green fl uorescent protein (GFP) or luciferase (Luc) was below –25 mV at the phys-iological pH condition ( Figure 1 b). The mineralization of Ad5 was achieved via a two-step process (Figure 1 a): the calcium-rich treatment of viral surfaces in normal saline (NS) afforded the calcium-rich superfi cial layers, which served as the nuclea-tion sites. [ 24 ] The desired mineralized Ad5 hybrid nanoparticles (named Ad5-CaPi) were then obtained with the utilization of a

bH & Co. KGaA, Weinheim wileyonlinelibrary.com 1

http://doi.wiley.com/10.1002/adhm.201200034

http://doi.wiley.com/10.1002/adhm.201200034

www.MaterialsViews.com

2

CO

MM

UN

ICATI

ON

www.advhealthmat.de

Figure 1 . Distinct physicochemical and biological characteristics of mineralized virus: a) Schematic presentation of in situ mineralization of Ad5 with core-shell structure; b) Zeta potential of Ad5; c) Direct TEM observation of Ad5-CaPi without stain treatment. (scale bar: 100 nm); Insert is Ad5-CaPi in high magnifi cation (scale bar: 50 nm); d) Energy-dispersive X-ray spectroscopy (EDS) analysis of Ad5-CaPi; e) X-ray diffraction (XRD) and f) Fourier transform infrared (FTIR) spectroscopy of Ad5-CaPi; g) Ad5 capsid protein hexon was identifi ed by dot blot assay under native or denatured condition with anti-hexon monoclonal antibody. V, Ad5; VCaPi, core-shell Ad5-CaPi; VP, Ad5 and CaPi co-precipitates.

controlled titration of dibasic sodium phosphate solution into the above calcium-modifi ed Ad5 suspension. Ad5 in the hybrid composites was identifi ed by the western blot assay and PCR assay (Figure S1, Supporting Information). The resulting core-shell like Ad5-CaPi possessed unique physical and chemical properties, as compared with the native Ad5. Under ambient

© 2012 WILEY-VCH Verlag Gwileyonlinelibrary.com

conditions the hybrid was completely covered by CaPi mineral, which was supported by the energy dispersive X-ray spectroscopy (EDS) measurement. Only the inorganic elements, Ca together with P and O of CaPi mineral phase were observed, whereas the organic components of the native virus were not detect-able (Figure 1 d). Moreover, it was proved that the mineralized

mbH & Co. KGaA, Weinheim Adv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201200034


CO

MM

UN

ICATIO

N

www.advhealthmat.de

CaPi shared the same properties of the typical amorphous cal-cium phosphate nanoparticles, as evidenced by the physico-chemical characterizations such as X-ray Diffraction (XRD) and Fourier transform infrared (FTIR) (Figure 1 e and 1 f).

Under transmission electron microscopy (TEM), the Ad5-CaPi exhibited homogenous spheres with typical diameter of 80 ± 5 nm, which was a little larger than that of the native Ad5 due to the mineral exterior (Figure 1 c and inset). It is worth noting that, owing to the change of surface repulsive force, Ad5-CaPi could be collected by low-speed concentration and observed directly under TEM without any stain treatment, which is not feasible for the native Ad5 (Figure 1 c and Figure S2). Hence, the concentration and characterization procedures are simplifi ed, benefi cial for the large-scale applications of such viral particles.

Moreover, due to the shielding effect of CaPi, Ad5-CaPi could prevent the recognition of Ad5 by its specifi c antibody under the extracellular conditions. As demonstrated by the Dot blot assay, Anti-hexon antibody could not determine the stealthy shielded Ad5 capsid protein inside the shell (Figure 1 g, VCaPi). By contrast, we also performed the control experiment in which simple mix of Ad5 with CaPi supersaturated solutions by normal co-incorporation methods. [ 25 ] The intact shell on the viral surface could not be fully warranted, leading to the detect-able of virus capsid (Figure 1 g, VP). It is evident that the cur-rent BVSE method exerted a signifi cant biological shielding effect for the living virus under native conditions.

On the other hand, in a denatured environment with pH around 5, the unstable CaPi phases could degrade automatically (Figure 1 g denatured condition), which arouse our interest to investigate whether or not the CaPi shell of Ad5-CaPi could be demineralizated and therein release the infectious virus under the slightly acidic intracellular conditions. HEK293 cells were chosen as the model cell to evaluate the release behavior as well as the infectivity recovery of virus-CaPi nanohybrids, as moni-tored by the fl uorescent signals derived from the Ad5-GFP. Based on our experiments, it is obvious that the shelled virus was spontaneously released from the hybrids after 12 to 24 h incubation, the mineralized virus, Ad5-GFP-CaPi, exhibited more intensive expression of GFP than the native counterpart ( Figure 2 a) (Figure S3 and S4). Furthermore, the quantitative release of Ad5 from the hybrid could also be achieved with the utilization of Ad5-Luc-CaPi, as refl ected by the cytopathic effect (Figure S5) and enhanced Luc expression at different multi-plicity of infection (MOI) (Figure 2 b). Such results indicated under extracellular conditions Ad5-CaPi exhibited as inanimate mineral, whereas after cellular uptake the acidic pH condition ensures the restore of animate virus. Therefore, different from other surface engineering, the biomineralization and subse-quent demineralization of the CaPi shell provided virus a con-trolled reversible transition between “living” and “non-living” conditions.

To our surprise, the biomineralization treatment could enhance the infection ability based on the above intracellular experiments as well as the in vivo experiments via intravascular injection (i.v.). For the comparison, BALB/c mice were intra-venously injected with Ad5-Luc and Ad5-Luc-CaPi respectively (Figure 2 c and 2 d). In vivo imaging exhibited that Luc produc-tion of Ad5-Luc peaked on day 4 (13.65 × 10 6 photons s − 1 cm − 2 sr − 1 ) and decreased on day 7 (1.31 × 10 6 photons s − 1 cm − 2 sr − 1 ),

© 2012 WILEY-VCH Verlag GmAdv. Healthcare Mater. 2012, DOI: 10.1002/adhm.201200034

with a duration of 10 days. In contrast, the Luc activity of Ad5-Luc-CaPi was observed as early as 6 h post injection (6.74 × 10 6 photons s − 1 cm − 2 sr − 1 ), peaked at day 4 (85.96 × 10 6 photons s − 1 cm − 2 sr − 1 ), and eliminated at day 15. Hence, The Ad5-Luc-CaPi gave rise to 6.3 fold higher Luc production as well as 5 days prolonged duration than the native Ad5-Luc, suggesting the signifi cant enhancement for both intensity and duration time. The effect of hybrid stability in serum could be fully excluded, as supported by the in vitro tolerability for 10 days incubation (Figure S6). These results indicate the Ca 2 + , dissolving from CaPi shells in the acidic endosome, increased the endosomal release by destabilization of its membrane. [ 26 ] It has also found that the increasing Ca 2 + can improve the cellular nuclear uptake of viral DNA through the nuclear pore complex. [ 14 , 27 ] In conse-quence, the CaPi phase dramatically stimulates the transfection effi ciency and long term expression with a relatively lower viral input dose. Moreover, the reason for earlier observation of Luc signal than that of native Ad5 is that Ca 2 + enhancement rise the intensity of gene expression, leading to detection of the signal within a shorter time. [ 28 ]

Next, we investigate the cellular uptake pathway for the Ad5 hybrid nanoparticles. As we know, native Ad5 adopt a CAR-dependent pathway to enter the cell, which was confi rmed by the phenomenon that Ad5-GFP failed to establish infection and express GFP in CAR defi cient cell lines (K562, NIH3T3 and CHO) ( Figure 3 a, 3 d, 3 g). However, in such CAR defi cient cells, Ad5-GFP-CaPi resulted in the effective GFP expression, implying that the mineral CaPi modifi ed the uptake as a CAR-independent pathway (Figure 3 b, 3 e, 3 h). In detail, we examine the Luc expression of Ad5-Luc-CaPi in time course assay. It is observed that Luc expression of Ad5-Luc-CaPi peaked at 24 h in K562, NIH3T3 and 48 h in CHO cells, which displayed 200-fold higher Luc intension than that of Ad5-Luc (Figure 2 c, f, i). As report, CaPi nanoparticles with diameters of ∼ 100 nm access to uptake via receptor-independent endocytic pathway. [ 15 , 28 ] The size of the prepared Ad5-CaPi nanohybrid is fully in according with such range and meets the cellular endocytosis pathway (Figure S8). It is suggested that the intrinsic biophysicochemical characteristics of inorganic phase (size, roughness, hydropho-bicity and charge, etc.) have a huge effect to infl uence the cel-lular uptake. Therefore, together with the mediation of size and mineral phase, the proposed BVSE method enables the virus hybrid to utilize a Trojan-horse like cellular entry process, which solves the receptor restriction during cellular transfection.

Moreover, we gain more insights into the organ distribu-tion of such hybrid nanoparticles. Based on the ex vivo tissue localization experiments of Ad5-Luc and Ad5-Luc-CaPi, both of them identifi ed the Luc production in abdomen before excise of organs at the 4th day post i.v. (Figure 3 j). But they also showed some differences. Liver was the major target organ for the Ad5-Luc (Figure 3 k, second panel), since Ad5-Luc circulated through blood stream to CAR positive hepatocytes. [ 22 ] In con-trast, for Ad5-Luc-CaPi, liver expression was reduced while lung and spleen expression were signifi cantly increased (Figure 3 k, third panel). Although the ex vivo results were not quantita-tive analyses considering the quenching effect of fl uorescent Luc during excised treatment, they indeed demonstrated quali-tatively that the CaPi shell ablated the natural tissue tropism of Ad5. We speculated that such expanded tissue transfection

3bH & Co. KGaA, Weinheim wileyonlinelibrary.com


4

CO

MM

UN

ICATI

ON

www.advhealthmat.de

Figure 2 . Gene transduction kinetics of mineralized virus in vitro and in vivo: a) Infectivity recovery and spontaneously release of Ad5-CaPi in HEK293 cells were indicated by expression of GFP; b) Growth kinetics of Ad5-Luc and Ad5-Luc-CaPi by RLU/mg protein in 12, 24, 48, and 72 hours with MOI of 1, 10, and 100, respectively; c) In vivo kinetics of Luc expression after intravenous injection with Ad5-Luc (left) and Ad5-Luc-CaPi (right), Luc production was imaged at day of 0, 2, 4, 6, 7, 8, 10, 12, 15; d) The capacity of Luc production was shown by bioluminescence intensity (Photons s − 1 cm − 2 sr − 1 ).

was coincident with the distribution features of pure nano-CaPi vectors. [ 29 ] The phenomena indicated that tropism expansion of virus hybrid in mice was attributed to the effect of mineral shell, which proved BVSE to be a novel technique for viral traffi c into different tissues with improved infectivity, benefi icial for the expansion of virus-based therapeutic applications.

Another important obstacle for the administration of viral vector is the pre-existing immunity, which should be taken into account for the further applications of the Ad5-CaPi hybrid. It is not unexpected that, for the native Ad5-GFP, the in vitro GFP expression in HEK 293 cells was remarkably blocked by anti-Ad5 serum ( Figure 4 a, Ad5), indicating that the pre-existing anti-

© 2012 WILEY-VCH Verlag Gwileyonlinelibrary.com

Ad5 antibodies suppress the infection of the virus. However, the infection of mineralized Ad5 hybrid was not neutralized under the same conditions (Figure 4 a, Ad5-CaPi). Moreover, in vivo experiments also revealed such distinct differences. In par-ticular, for the Ad5 pre-immunized mice, Luc was completely absent after the administration of Ad5-Luc. In contrast, admin-istration of virus-mineral hybrid resulted in the strong Luc expression, peaking at day 4 (82.61 × 10 6 photons s − 1 cm − 2 sr − 1 ), declining at day 6 in a dosage of 1.09 × 10 9 VP/ml, and lasting for 9 days (Figure 4 b and 4 c). Hence, both in vitro and in vivo results strongly demonstrated that the mineralized virus could overcome the pre-existing immunity and infect effi ciently, even



CO

MM

UN

ICATIO

N

www.advhealthmat.de

Figure 3 . Receptor independent infection in CAR-defi cient cell lines and tropism expansion of mineralized virus: Cellular entry of virus-CaPi and native virus were observed by expression of GFP and quantifying expression of Luc in myeloid-derived cell lines K562 (a, b and c); rodent cells NIH3T3 (d, e and f) and CHO (g, h and i) at 50 MOI at 24 h or 24 h to 72 h. All data were expressed as RLU/mg protein. j) In vivo imaging of Ad5-Luc (left) and Ad5-Luc-CaPi (right) after i.v.at day of 4. k) Organ distributions of NS control (fi rst panel), Ad5-Luc (second panel) and Ad5-Luc-CaPi (third panel) by ex vivo imaging.

in the environment of neutralizing antibody. It is supposed that the high coverage ratio of mineral shell enables the stability and protection during the virus delivery under physiological pH. As


a result, antibody inactivation is considerably suppressed; this is attributed to the ablation of direct recognition between virus capsid protein and biological viral trap. Therefore, due to the



CO

MM

UN

ICATI

ON

www.advhealthmat.de

Figure 4 . Protection effects of mineral shells against pre-existing anti-Ad5 antibodies. a) Ad5-GFP and Ad5-GFP-CaPi were pre-incubated with normal mice serum or anti-Ad5 serum and applied to HEK 293 cells, respectively. GFP expressions were observed using fl uorescent microscopy. b) Pre-immunized mice were intravenous injected with Ad5-Luc (left) and Ad5-Luc-CaPi (right), and Luc production was tested by in vivo imaging at day 1, 2, 4, 6, and 9, respectively. c) The capacity of Luc production was shown by bioluminescence intensity.

protective ability of CaPi shell, the hybrid nanoparticles could serve as potential therapeutic vectors, advantageous for the repeating administrations in pre-immunized human popula-tions in high-prevalence areas.

6 © 2012 WILEY-VCH Verlag Gwileyonlinelibrary.com

It should be noted that, other kinds of virus with therapeutic or diagnostic potentials, such as Japanese Encephalitis Virus (JEV) vaccine strain, etc., could also be handled with BVSE to solve the similar problems as Ad5 (Figure S7). Although the



CO

MM

UN

ICATIO

N

www.advhealthmat.de

surface structure varies from capsid virus (Ad5) to envelope virus (JEV), the universality of BVSE strategy is mainly based on the strong negative charge on the viral surfaces, [ 9 ] which can enrich calcium ions readily under certain solution conditions. Therefore, the BVSE are cost-effective, easy to perform and universal to the fabrication of virus-inorganic complex without additional genetic or chemical tailoring.

In summary, the universal BVSE technique affords a simple, rapid, and versatile strategy to fabricate the artifi cial viral core-shell hybrid by means of biomineralization, which signifi cantly modifi es the chemical, physical and biological properties of the virus and is benefi cial for virus reprocessing and protection. The virus hybrids with CaPi attributes stealthily bypass the receptor barriers with enhanced infectivity and expanded tro-pism. Moreover, they can circumvent neutralizing antibodies, ensuring systemic administration of the virus under the pre-immunized conditions in relatively low administrating dosage. Therefore, the BVSE technique overcomes the limitations of the native virus and opens up the multiple applications of virus with therapeutic potential. Furthermore, such a material-based incorporation with virus provides an effi cient strategy to develop viral materials with the characteristics of functional shells.

Supporting Information Supporting Information is available from the Wiley Online Library or from the author.

Acknowledgements We thank Hua Wang (Beijing Institute of Radiation Medicine, Beijing) and Allbringt Biotech (Shanghai) for providing Ad5-GFP and Ad5-Luc viruses. We thank Peiyong Shi (Novartis Institute for Tropical Diseases, Singapore) and Feng Wang (University of Science and Technology of China) for their critical reading of the manuscript. We are also grateful to Guang Tian, Ge Yan and Yuchuan Li for their technical assistances in TEM characterizations. This work was supported by the Fundamental Research Funds for the Central Universities (Zhejiang University KYJD09031), the National Natural Science Foundation of China (No.20871102 and No.30972613), and Daming Biomineralization Foundation. Chengfeng Qin was supported by the Beijing Nova Program of Science and Technology (No.2010B041).

Received: December 29, 2011 Revised: February 8, 2012

Published online:

[ 1 ] T. Douglas , M. Young , Adv. Mater. 1999 , 11 , 679 . [ 2 ] T. Douglas , M. Young , Science 2006 , 312 , 873 .


[ 3 ] C. E. Thomas , A. Ehrhardt , M. A. Kay , Nat. Rev. Genet. 2003 , 4 , 346 . [ 4 ] S. K. Campos , M. A. Barry , Curr. Gene Ther. 2007 , 7 , 189 . [ 5 ] F. Kreppel , S. Kochanek , Mol. Ther. 2008 , 16 , 16 . [ 6 ] P. S. Banerjee , P. Ostapchuk , P. Hearing , I. Carrico , J. Am. Chem.

Soc. 2010 , 132 , 13615 . [ 7 ] Z. L. Xu , H. Mizuguchi , F. Sakurai , N. Koizumi , T. Hosono ,

K. Kawabata , Y. Watanabe , T. Yamaguchi , T. Hayakawa , Adv. Drug. Deliv. Rev. 2005 , 57 , 781 .

[ 8 ] P. N. Reynolds , S. A. Nicklin , L. Kaliberova , B. G. Boatman , W. E. Grizzle , I. V. Balyasnikova , A. H. Baker , S. M. Danilov , D. T. Curiel , Nat. Biotechnol. 2001 , 19 , 838 .

[ 9 ] X. Wang , Y. Deng , H. Shi , Z. Mei , H. Zhao , W. Xiong , P. Liu , Y. Zhao , C. Qin , R. Tang , Small 2010 , 6 , 351 .

[ 10 ] D. N. Nguyen , J. J. Green , J. M. Chan , R. Langer , D. G. Anderson , Adv. Mater. 2009 , 21 , 847 .

[ 11 ] E. Dujardin , S. Mann , Adv. Mater. 2002 , 14 , 775 . [ 12 ] B. Wang , P. Liu , R. Tang , Bioessays 2010 , 32 , 698 . [ 13 ] W. Shenton , T. Douglas , M. Young , G. Stubbs , S. Mann , Adv. Mater.

1999 , 11 , 253 . [ 14 ] A. Maitra , Expert. Rev. Mol. Diagn. 2005 , 5 , 893 . [ 15 ] D. Jiang , G. S. Premachandra , C. Johnston , S. L. Hem , Vaccine 2004 ,

23 , 693 . [ 16 ] T. T. Morgan , H. S. Muddana , E. I. Altinoglu , S. M. Rouse ,

A. Tabakovic , T. Tabouillot , T. J. Russin , S. S. Shanmugavelandy , P. J. Butler , P. C. Eklund , J. K. Yun , M. Kester , J. H. Adair , Nano Lett. 2008 , 8 , 4108 .

[ 17 ] B. Wang , P. Liu , W. Jiang , H. Pan , X. Xu , R. Tang , Angew. Chem. Int. Ed. Engl. 2008 , 47 , 3560 .

[ 18 ] P. L. Leopold , R. G. Crystal , Adv. Drug. Deliv. Rev. 2007 , 59 , 810 . [ 19 ] J. A. St George , Gene. Ther. 2003 , 10 , 1135 . [ 20 ] M. C. Bewley , K. Springer , Y. B. Zhang , P. Freimuth , J. M. Flanagan ,

Science 1999 , 286 , 1579 . [ 21 ] N. Belousova , V. Krendelchtchikova , D. T. Curiel , V. Krasnykh , J.

Virol. 2002 , 76 , 8621 . [ 22 ] S. N. Waddington , J. H. McVey , D. Bhella , A. L. Parker , K. Barker ,

H. Atoda , R. Pink , S. M. Buckley , J. A. Greig , L. Denby , J. Custers , T. Morita , I. M. Francischetti , R. Q. Monteiro , D. H. Barouch , N. van Rooijen , C. Napoli , M. J. Havenga , S. A. Nicklin , A. H. Baker , Cell 2008 , 132 , 397 .

[ 23 ] D. M. Roberts , A. Nanda , M. J. Havenga , P. Abbink , D. M. Lynch , B. A. Ewald , J. Liu , A. R. Thorner , P. E. Swanson , D. A. Gorgone , M. A. Lifton , A. A. Lemckert , L. Holterman , B. Chen , A. Dilraj , A. Carville , K. G. Mansfi eld , J. Goudsmit , D. H. Barouch , Nature 2006 , 441 , 239 .

[ 24 ] P. Atanasova , D. Rothenstein , J. J. Schneider , R. C. Hoffmann , S. Dilfer , S. Eiben , C. Wege , H. Jeske , J. Bill , Adv. Mater. 2011 , 23 , 4918 .

[ 25 ] A. Fasbender , J. H. Lee , R. W. Walters , T. O. Moninger , J. Zabner , M. J. Welsh , J. Clin. Invest. 1998 , 102 , 184 .

[ 26 ] T. Shiraishi , S. Pankratova , P. E. Nielsen , Chem. Biol. 2005 , 12 , 923 . [ 27 ] U. F. Greber , M. Suomalainen , R. P. Stidwill , K. Boucke ,

M. W. Ebersold , A. Helenius , EMBO J. 1997 , 16 , 5998 . [ 28 ] R. E. Dolmetsch , K. Xu , R. S. Lewis , Nature 1998 , 392 , 933 . [ 29 ] I. Roy , S. Mitra , A. Maitra , S. Mozumdar , Int. J. Pharm. 2003 , 250 , 25 .


Biomineralization-Based Virus Shell-Engineering: Towards Neutralization Escape and Tropism Expansion

Documents

Transcript of Biomineralization-Based Virus Shell-Engineering: Towards Neutralization Escape and Tropism Expansion