www.rsc.org/materialsRegistered Charity Number 207890

Showcasing research on mesoporous protein thin

fi lms for molecule delivery from the Peng’s Lab at the

Zhejiang University.

Title: Mesoporous protein thin fi lms for molecule delivery

Large scale mesoporous free-standing protein thin fi lms were

prepared through a simple fi ltration technique using metal

hydroxide nanostrands as frames and pore templates. The porous

protein fi lms could recyclably deliver molecules effi ciently

stimulated by pH.

As featured in:

See H. Huang et al.,

J. Mater. Chem., 2011, 21, 13172.

0959-9428(2011)21:35;1-F

ISSN 0959-9428

www.rsc.org/materials Volume 21 | Number 35 | 21 September 2011 | Pages 13081–13684

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FEATURE ARTICLEXiaogang Liu et al.Emerging functional nanomaterials for therapeutics

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Dynamic Article LinksC<Journal ofMaterials Chemistry

Cite this: J. Mater. Chem., 2011, 21, 13172

www.rsc.org/materials PAPER

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Mesoporous protein thin films for molecule delivery†

Hongwen Huang, Qing Yu, Xinsheng Peng* and ZhiZhen Ye

Received 13th March 2011, Accepted 16th May 2011

DOI: 10.1039/c1jm11090j

A simple and easy method was developed for preparation of large-scale free-standing mesoporous

protein thin films through a filtration technique by using ultrathin metal hydroxide nanostrands as

frames and pore templates. Composite nanofibrous dispersions of nanostrands and proteins were

formed by assembling negatively charged protein on the highly positively charged nanostrand surfaces.

These dispersions were filtered on a porous substrate. After partially cross-linking the amino groups of

the proteins, removing the nanostrands and peeling off from the substrate, free-standing mesoporous

protein films were obtained. These films repeatedly demonstrated efficient loading and releasing

performance of dye molecules by pH controlling. The delivery capacity was 33.5% relative to the weight

of the matrix. This value is several times higher than that of active carbon powders as well as that of

metal hydroxide sludge. At pH lower than the isoelectic point of protein, the negatively charged dye

molecules were loaded. Subsequently, the preloaded dye molecules were released at pH higher than the

isoelectric point of protein. Furthermore, reversible delivery of doxorubicin drug molecules was

realized by using these mesoporous protein thin films under physiological conditions. These films hold

promising applications for recovering dyes from the dye waste water, and for efficient drug delivery

platforms with controllable releasing speed.

1 Introduction

Controlling the delivery of molecules, especially drug molecules,

is very important for biomedical applications.1–3 Except for the

most popular hydrogel systems,4–8 porous nanomaterials are the

next most desirable systems for these purposes. Microcapsules,

carried with well pre-designed nanochannels or nanovalves have

been investigated for controlling drug delivery.9–16 Although the

carriers show great promise for cancer applications, critical

limitations persist. Many synthetic materials suffer from

biocompatibility and toxicity issues.17 Therefore, protein-based

materials are attracting much more attention and are ideal

platforms for drug delivery due to their biocompatibility and

biodegradability as well as low toxicity.17 Various proteins have

been reported for drug delivery systems, including soy, gelatin,

apoferritin, heat shock protein, etc.18–24 Especially, protein cages

are promising for drug loading and releasing systems, since they

can offer different surfaces and a variety of chemical and bio-

logical structures for drug delivery. Natural stable protein cages

in most physiological environments help to protect drugs from

enzymatic degradation. Zheng’s group engineered high density

State Key Laboratory of Silicon Materials, Department of MaterialsScience and Engineering, Zhejiang University, Hangzhou, 310027, P. R.China. E-mail: pengxinsheng@zju.edu.cn; Fax: +86-571-87952625; Tel:+86-571-89751958

† Electronic supplementary information (ESI) available: TEM images,the preparation and the molecular delivery results of the non-porousprotein films. See DOI: 10.1039/c1jm11090j

13172 | J. Mater. Chem., 2011, 21, 13172–13179

lipoprotein-based nanocarriers for enhancing cancer target

delivery.25 Most of the above works were investigated in the form

of separated protein capsules or nanoparticles. Assembling these

protein-based capsules into the form of robust thin films make

some advantages for easy handling, recovery and reusage.17,21

However, it is difficult to build robust thin films by post-

assembling the natural (or pre-synthesized) micro or nano-

capsules without blocking their channels or valves. Recently,

cross-linked soy protein films have been prepared and shown

kinetic delivery of drugs. The drug molecules were incorporated

into the matrix in situ during the film preparation process and

released by erosion from the matrix.21 The drawbacks of this

method are that the drugs may lose their activity during the

cross-linking process, and the films cannot be reused after

releasing the drugs.

Apoferritin contains 14 channels and one cage (12 nm outer

diameter and 8 nm inner diameter) for exchanging its cargo

between the cage’s interior and exterior environments.26 Based

on this feature, apoferritin has been widely used for drug delivery

systems17,22–24 as well as the preparation of uniform nano-

particles.27–29 However, due to the small diameters of the chan-

nels (3–4 �A),30 only small ions or molecules could entrap into (or

release out of) its cage.31,32 This limitation is an obstacle for its

application in the delivery of many drug molecules with diame-

ters of 2–3 nm, for example, doxorubicin, a general anticancer

drug. To overcome this limitation, some researchers prepared an

apoferritin based drug delivery system by disassembling the

apoferritin cages into subunits at pH 2, allowing the dispersed

This journal is ª The Royal Society of Chemistry 2011

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drugs to load, and reassembling into apoferritin cages when the

pH was changed to basic pH 8.5.31,32 However, the releasing of

these relatively big molecules through the apoferritin narrow

channels is still a challenge.

In order to build porous networks with desirable diameters,

based on our previous work on the preparation of an ultrathin

ferritin water separation membrane process,33 in the present

work we report a simple method to prepare large scale free-

standing mesoporous protein films through filtration techniques

by using ultrathin (2.5 nm in diameter) metal hydroxide nano-

strands34 as frames and pore generators. After partially cross-

linking the amino groups of the protein by glutaraldehyde,

removing away the nanostrands and peeling away from the

substrate, free-standing mesoporous apoferritin films were

obtained. These films demonstrated efficient loading and

releasing of dye molecules stimulated by pH. The loading

capacity was 33.5% relative to the weight of the matrix. This was

5.0 times that of metal hydroxide sludge,35 and 3.7 times that of

active carbon powders.36,37 When the pH value was less than the

isoelectric point (Ip) of apoferritin, negatively charged dye

molecules were loaded into the positively charged protein film

through the mesochannels. Subsequently, the preloaded dye

molecules were released at pH higher than the Ip value.

Furthermore, delivery of doxorubicin drug molecules was also

realized by using these apoferritin thin films. These films hold

potential applications both for recovering dyes from dye waste

water and for controlling drug delivery.

2 Experimental

2.1 Materials

Copper nitrate (Cu(NO3)2�2.5H2O, aminoethanol

(NH2CH2CH2OH), HCl, NaOH and glutaraldehyde were

purchased from Across Chemical. Direct yellow 50 (DY), Evan

blue (EB), doxorubicin (Dox), apoferritin and ferritin were

purchased from Sigma-Aldrich and used without further purifi-

cation. A polycarbonate (PC) membrane (Whatman) and anodic

porous alumina membrane (Whatman) with pores of 200 nm,

and effective diameter of 3.2 cm were used for the preparation of

the films. Deionized water (18.2 MU) was produced by a Milli-

pore Direct-Q System, and used throughout the experiments.

2.2 Protein films

The nanostrands solution was prepared by mixing 5 ml aqueous

copper nitrate (4 mM) with an equal volume of 1.6 mM ami-

noethanol solution under vigorous stirring and aging for 2 days.

Typically, the free-standing apoferritin films were prepared by

mixing 0.75 ml, 0.38 mg ml�1 protein (apoferritin or ferritin)

solution with the copper hydroxide nanostrands solution under

stirring for 15 min to form a composite nanofibrous dispersion

by electrostatic interaction. A filter cake was formed by filtering

the mixture solution on a porous PC membrane with pore size of

200 nm and effective diameter of 3.2 cm under 80 kPa pressure.

The proteins in the filter cake were cross-linked by filtering 5 ml,

5 wt% glutaraldehyde solutions for 10 min. This was very

different from that for ultrafast water purification protein

membranes by using 25 wt% glutaraldehyde, and cross-linking

for 2–3 h.33 After washing away the remaining cross-linkers, the

This journal is ª The Royal Society of Chemistry 2011

film was peeled off from the PC membrane surface in ethanol.

Free-standing mesoporous protein films were obtained by

removing away the nanostrands by 10 mM HCl solution.

2.3 Dye molecule delivery

For dye loading, one piece of the prepared 3.2 cm protein film

was immersed into 10 ml, 10 mM DY (or EB) dye solution with

pH less than 5.5. The loading process was monitored by Uv-vis

spectroscopy. The Uv-vis spectra of the solution were recorded

after certain immersing time intervals. The loading amount was

calculated relative to the original dye concentration. The

releasing process was examined by immersing the protein film

with loaded dye into 10 ml pure water with different pH starting

from 5.53. The releasing amount was calculated from the Uv-vis

spectra which were recorded from the dye solutions at certain

time intervals. The pH of the solution was adjusted by using HCl

and NaOH solutions.

2.4 Drug loading and releasing

Similar to the dye molecule loading and releasing process, one

piece of the prepared 3.2 cm protein film was immersed into 10

ml, 10 mM Dox aqueous solution at pH 6.6 (similar to the

physiological pH of human body fluid). The loading process was

monitored by Uv-vis spectroscopy. The Uv-vis spectra of the

solution were recorded after certain immersing time intervals.

The loading amount was calculated relative to the original Dox

concentration. The releasing process was examined by immersing

the Dox loaded protein film into 20 ml pure water with pH 5.0

close to the tumor surrounding environment pH value. The

releasing amount was calculated from the Uv-vis spectra of the

Dox concentration in the solutions after certain time. The pH of

the solution was adjusted by HCl and NaOH solutions.

2.5 Characterization

The morphologies and structures of the films were characterized

by using scanning and transmission electron microscopes (SEM,

Hitachi S-4800 and TEM, CM 200UT, Philips). SEM observa-

tion was conducted after coating a 2 nm thick platinum layer by

using a Hitachi e-1030 ion sputter at a pressure of 10 Pa and

a current density of 10 mA. UV-vis absorption spectra were

obtained by using a SHIMAZU UV-3600 spectroscopy instru-

ment. All the measurements were carried out under atmospheric

conditions. The apoferritin film was etched by Ar plasma with

power 10 W, 0.5 Pa pressure at room temperature for 3 min.

3 Results and discussion

3.1 Morphology and structures

The surface morphology of the prepared apoferritin film is

shown in Fig. 1a. The globular protein particles are clearly seen

and packed. Among them, some voids exist. The cross-section

SEM image of the film was recorded after being transferred on an

anodic alumina oxide membrane surface. The thickness of the

film was about 300 nm as shown in Fig. 1c. In order to see the

detail of the pore structures, the prepared apoferritin film was

etched by Ar plasma at 10 W and 0.5 Pa for 3 min at room

J. Mater. Chem., 2011, 21, 13172–13179 | 13173

Fig. 1 SEM images of 300 nm apoferritin film: (a) surface view, (b) low

and (c) high magnification cross-section view, (d) after etching by Ar

plasma at 10 W, 0.5 Pa for 3 min.

Scheme 1 The molecular structures of DY, EB, and Dox, respectively.

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temperature. After that, the surface was investigated by SEM as

shown in Fig. 1d. Plenty of linear grooves were seen and resulted

in porous networks. The diameter of these grooves is about

3–5 nm, which is slightly larger than that of the nanostrands

diameter, 2.5 nm. It is obvious that these grooves were generated

by the duplication of the nanostrands. These results show that

a very porous mesoporous matrix was really formed by the

process of filtrating, cross-linking and removing away the

nanostrands. The TEM images (see ESI, Fig. S1†) indicate that

the nanostrands are completely removed by HCl treatment. Plus,

the multifunctional properties of apoferritin groups, such as

porous, flexible, free-standing thin films provide a desirable

platform for molecular delivery systems.

3.2 Dye molecule loading and releasing

The prepared apoferritin film was robust enough for repeatedly

handling and examining the molecule delivery process at pH in

the range of 1.5 to 12.08.

It is well known that the charge properties of protein

remarkably depend on the pH value. When the pH is higher than

its Ip point, the protein will be negatively charged. Otherwise, if

the pH is lower than its Ip value, the protein will be positively

charged.38

The Ip of apoferritin (or ferritin) is at pH 4.5–4.8.26,33 When

the pH is higher than this value, the apoferritin film will be

negatively charged. This negatively charged film could attract

and load positively charged ions or molecules from the solution

through electrostatic interactions, and vice versa. Subsequently,

the preloaded negatively charged molecules could be released at

pH higher than the Ip point. Of course, at higher pH, the pre-

loaded positively charged molecules could be released at pH less

than the Ip point. These properties are desirable for reversible

delivery of molecules by pH controlling. Negatively charged DY

molecules were first examined. The loading and releasing mole-

cules were monitored by UV-vis spectroscopy.

The molecular structure of DY is shown in Scheme 1. Fig. 2a

shows the loading procedure of 10 ml, 10 mM DY dye solution

with pH 1.5 by using a 300 nm thick apoferritin film with

13174 | J. Mater. Chem., 2011, 21, 13172–13179

diameter of 3.2 cm. It is clear that the intensity of the charac-

teristic absorption peak of DY at 400 nm decreased with the

elongation of time. This indicates that negatively charged DY

molecules were loaded by the positively charged apoferritin film.

The loading behaviors of the protein film under different pH are

shown in Fig. 2b. It is clear that the lower the pH, the more DY

molecules are loaded, and the faster the speed is. At pH 1.50, 21%

dye was loaded within 10 min, and finally 99.6% dye molecules

were loaded after 7 h. However, at pH 4.5, only 17.5% DY

molecules were loaded for 23.7 h. At pH 5, almost no loading was

observed even for 2 days (not shown). This pH value was slightly

higher than the Ip point of the native apoferritin. After loading

DY at 1.5 pH for 7 h, the protein film was immersed into 10 ml

water with different pH, starting from pH 5.53 for releasing the

pre-loaded DYmolecules. The results are shown in Fig. 2c. It can

be seen that almost no DY molecules are released from the

protein films at pH 5.53 for 5 h or more. By increasing the pH to

6.74, DY molecules are slowly released up to 12% for 5 h. The

higher the pH, the quicker the releasing speed is, and the more

the amount of dye molecules that are released. At pH 12.08,

51.5% dye molecules are released within 1 min, and 85.5% dye are

released after 7 h. Fig. 2d shows the releasing amount of DY for 1

min at different pH. At the initial stage, the more negatively

charged the protein matrix, the faster the releasing speed is. The

photo images of the original protein film, loaded DY at pH 1.5

and after releasing DY at pH 12.08 are shown in Fig. 2e, 2f,

and 2g, respectively. These images further confirmed that the

apoferritin film could efficiently deliver dye molecules stimulated

by pH.

The loading and releasing processes were performed repeat-

edly. Fig. 3 shows the performance of the apoferritin film

repeatedly loading and releasing DYmolecules. Significantly, the

loading capacity of the film is recovered after releasing the dyes.

The releasing efficiency was increased from 85.6% in the first

cycle to 99.6% in the next cycle. 14.4% dye molecules remained in

the matrix after the first releasing process, which might have been

trapped by the apoferritin cavities. However, after the first cycle,

almost all the reloaded dye molecules were released from the film.

This journal is ª The Royal Society of Chemistry 2011

Fig. 2 Loading and releasing of DY molecules by using a 300 nm apoferritin film: (a) UV-vis spectra of the loading process at pH 1.5 with time, (b) the

loading performances of DY under different pH, (c) releasing performances of DY in water under different pH, (d) the releasing amount within the first

one minute at different pH. And the photo images of (e) the original apoferritin film; (f) after immersing in 10ml, 10 mMDY at pH 1.5 for 6.5 h and (g)

after releasing DY into water at 12.08 pH for 2 h.

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The weight of the 99.6% loaded amount of DY was 0.0956 mg.

The apoferritin film weight was 0.285 mg. The weight of the

loaded dye molecules was 33.5% of that of the apoferritin film.

This value is 3.7 times that of active carbon powders,36,37 and 5.0

times that of metal hydroxide sludge.35 These indicate that most

of the DYmolecules were loaded into and released out of the film

through the porous networks, respectively.

The EB dye molecule has a similar shape to that of the DY

molecule, except that the EB molecule has two –NH2 groups.

The loading and releasing experiments of EB were carried out at

pH 1.5 and pH 12.09, respectively, as shown in Fig. 4. The

This journal is ª The Royal Society of Chemistry 2011

loading and releasing performance of EB was very close to that

of DY. 40% EB molecules were loaded into the apoferritin

matrix within 15 min. The loading amount reached up to 95%

for 3 h. The photo images are shown in Fig. 4b. The original

blue solution is remarkably discolored. And the protein films

become blue. All of these indicate that the EB molecules were

almost loaded by the protein film. When immersing the pre-

loaded EB apoferritin film into pH 12.09 water solution, the EB

molecules immediately released. The different color of the EB

solutions is due to the EB molecules changing their electron

structure at high basic pH. The dye molecules were efficiently

J. Mater. Chem., 2011, 21, 13172–13179 | 13175

Fig. 3 Reversibility of an apoferritin film for loading DY at pH 1.5

(black) and releasing of DY at pH 12.08 (red), respectively. The loading

time was 400 min, the releasing time was 260 min, respectively.

Fig. 4 (a) The loading (black) performances of EB by apoferritin films at

pH 1.5 and releasing (red) the preloaded EB at pH 12.09 during the first

cycle, respectively. (b) From left to right, the photo images of the original

EB solution at 1.5 pH; after immersing apoferritin for 1 day; the initial

releasing of EB in water at pH 12.09; and 2.5 h, respectively.

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transferred from one bottle to another bottle through the apo-

ferritin film.

3.3 Drug delivery

The above results show that the protein films could deliver dye

molecules by pH controlling. These results demonstrated that the

mesoporous apoferritin film could be applicable for dye waste

water treatment and recovering the dye molecules.

13176 | J. Mater. Chem., 2011, 21, 13172–13179

The biocompatibility of these protein films is wonderful for

drug delivery at physiological conditions. As mentioned before,

due to the small channel of apoferritin, reversible loading and

releasing of the drug is not possible with size larger than 0.4 nm.30

Robust porous apoferritin networks with pore diameter of about

3–5 nm (Fig. 1d) are desirable for reversible delivery of drugs

with diameters in this range. The additional advantage of the thin

film form is that it can be easily handled, recovered and reused.

Doxorubicin, a popular anticancer drug,39,40 with size of 2.6 �3.1 nm, as shown in Scheme 1, was examined. Since the drug is

normally active at physiological conditions, here, the experiment

for doxorubicin loading was carried out at pH 6.6, and the

releasing was carried out at pH 5.0 close to the tumor environ-

ment pH.40 The Uv-vis spectra, Fig. 5a, show that the concen-

trations of the drugs in the solution decreased with loading time.

The loading amounts are 17.8%, 28.7%, 39.8%, 54.8%, 66.6%,

74.1%, 83.6, 92.4% and 97.5% with loading times of 0.1, 0.2, 0.3,

0.6, 1.2, 2.4, 3.6, 6, and 8.4 h, respectively. The releasing speed is

much slower than that of the loading process. It took about two

days for the releasing of 90% Dox from the apoferritin matrix.

Quick loading and slow releasing are desirable for cancer treat-

ment.17 For drug delivery systems, keeping the drug activity is

most important. Fig. 5a and c show the same characteristic Uv-

vis peaks of the releasing Dox molecules as those of the original

Dox molecules, which indicates that the drug molecules reserved

their original activities.

3.4 Molecular delivery mechanism

From the structure of apoferritin, it is known that one apo-

ferritin has 664 amine (–NH2) units.41 The cross-linking reac-

tion took place between the amino groups and glutaraldehyde

molecules. The loading process occurred between the positively

charged amine groups and negatively charged sulfonate groups

of DY. One DY molecule has four sulfonate groups as shown in

Scheme 1. Because the distance between the amino groups of

the protein units is 0.36 nm,42 which is much smaller than that

between the sulfonate groups within one DY molecule (1.2 to

3.1 nm), the steric effects and electrostatic repulsion between the

DY molecules would not allow three or more sulfonate groups

of one DY molecule to be neutralized by the remaining amino

groups of apoferritin. One or two sulfonate groups of one DY

molecule bonded to the amino groups from the protein was

most reasonable. Since 99.6% DY were loaded into the protein

matrix, the mole ratio between DY and apoferritin was as high

as 160 (the molecular weight of DY is 956.8 and that of apo-

ferritin is 456000).26 If one or two sulfonate groups of DY were

neutralized by one amino group of apoferritin, the cross-linking

efficiency is in the range of 51.9 to 75.9, and resulting in robust

free-standing protein film. All of the amino groups were posi-

tively charged and completely neutralized by the negatively

charged DY. This analysis is also applicable to EB. The two

–NH2 groups of EB have no significant effect on the delivery

speed. The fitted curves of the loading and releasing process of

DY are shown in Fig. 6. They obey an exponential rule, eqn (1),

similar to the literatures reported about the releasing of liquid

pesticide from glutaraldehyde cross-linked sodium alginate

beads43 and a drug delivery system of a hydrophilic polymer

matrix44:

This journal is ª The Royal Society of Chemistry 2011

Fig. 5 Loading and releasing doxorubicin performance by using a 300 nm apoferritin film: (a) Uv-vis spectra of doxorubicin in the solution with

different loading time intervals and (b) the loading amount and time effect calculated from (a); (c) Uv-vis spectra recorded from the solution by releasing

doxorubicin in 20 ml water with pH of 5.0. The original doxorubicin solution is 10 ml, 10 mM, pH 6.6.

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Mt � 100% ¼ 1 � b0exp(c0D*t) (1)

Where Mt is the amount normalized to the original amount of

the molecules, b0, C0 are constants, D is the diffusion constant,

t is time. For the loading of DY at pH 1.5 as shown in Fig. 6a, b0and c0D are 0.97 and �0.0136, respectively. The correlation

coefficient R2 is 0.9975. For the releasing process, Fig. 6b, b0, c0D

and R are 0.46, �0.1522 and 0.9876, respectively. These results

demonstrate that the loading is quickly finished within a few

hours at lower pH with a higher postively charged protein matrix

through electrostatic interaction, and the releasing is also quick

at higher pH with a higher negatively charged protein matrix

through electric repulsion, respectively. The diffusion constant of

the releasing process is 11 times that of loading process. One

reason might be that during the loading process, the molecules

were first binded to the most outer surface of the pore, which will

slow down the next molecules entering into the deep channel.

However, during the releasing process, the molecules binded on

the most outer surface of the pore were released and gave way for

the inner molecules going out. Another reason for this is that the

protein matrix would expand at high pH such as 12.08 as we

described elsewhere.33

These behaviors are very different from the in situ co-assem-

bling and erosion process.21 The loading process is generated by

electrostatic attractive interactions. The interaction started from

the surface to the inner porous networks. A small amount of

about 14% DY were trapped in the apoferritin cavities due to the

partial disassembling of the apoferritin subunits at pH 1.5.31,32

This journal is ª The Royal Society of Chemistry 2011

But this amount could not be released at pH 12.08 due to the

reassembled apoferritin cavities at pH higher than 8.5 with small

channels in size of 3–4 �A,30–32 much smaller than the size of DY,

EB molecules (1.7 � 3.1 nm).33 For exploring this, ferritin with

fully filled iron oxyhydroxide cavities were used for preparing

a protein film with thickness of 300 nm by the same method and

conditions as those for preparation of apoferritin film. The DY

molecule loading and releasing performance of this ferritin film

was investigated. The results are shown in Fig. 7. It is clear that

almost all the dye molecules were released during the first cycle.

These results confirmed that the 14% unreleased DY molecules

were trapped into apoferritin cavities. However, after the first

cycle, all the reloaded DYmolecules in the following cycles could

be completely released out from the apoferritin matrix with

elongation time as shown in Fig. 3.

Different from the negatively charged DY and EB molecules,

Dox molecules are general positively charged at physiological

pH. Here, the loading pH of Dox is 6.6. At this pH value, the

apoferritin film is slightly negatively charged. Dox molecules

were diffused into the porous protein matrix and loaded by the

negatively charged sites slowly, as shown in Fig. 5a. The releasing

process is much slower than that of DY and EB. The reason is

that pH 5.0 is very close to the Ip point pH 4.5–4.8 of apoferritin

(ferritin). At this pH, the protein film is very slightly negatively

charged. The electrostatic interaction between Dox and the

protein matrix is very weak and broken slowly. The Dox mole-

cules were released and diffused out. The slow release rate of

drug is desirable for cancer treatment.17 If releasing speed is too

J. Mater. Chem., 2011, 21, 13172–13179 | 13177

Fig. 6 Fitted curves of loading DY at pH 1.5 and releasing DY at pH

12.08, respectively by exponential equation. The black is the original

data, the red is the fitted curves.

Fig. 7 (a) The loading performance of DY by ferritin and apoferritin

films at pH 1.50, respectively. (b) The corresponding releasing perfor-

mance of DY from ferritin and apoferritin at pH 12.08, respectively.

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fast, the local concentration of drug will be too high and induce

toxicity for the other cells.

To distinguish the role of the mesoporous channels for the

molecules delivery, the loading and releasing performances of the

non-porous protein film with 3.2 cm diameter and 0.285 mg

apoferritin were examined. These non-porous protein films were

prepared by direct filtration of apoferritin on the surface of

a nanostrands filter cake layer surface and cross-linking (see

ESI†). The results in Fig. S2 show that its loading capacity of DY

at pH 1.5 is about 19% for 10 h.Within 2 min, 17%DYmolecules

are loaded. After that the loading process is slower and slower.

The releasing process is very quick. 17.5% DY molecules are

released within 2 min. The remaining 1.5% are released very

slowly. 0.7% are still not released after 10 h. All the results

indicate that most of the DY were loaded on the outermost

surface of the non-porous protein film. The loading capacity for

10 h is 20% of that loaded by the mesoporous protein films

prepared with nanostrands as channel templates. The reason is

that, at the initial stage, DY molecules were immediately loaded

on the non-porous protein film surface by electrostatic interac-

tion. After that, it was very hard for the DYmolecules to go deep

13178 | J. Mater. Chem., 2011, 21, 13172–13179

into these protein films, since not so many channels existed there.

From this, it can be concluded that the mesoporous channels

generated by the nanostrands contribute more to the high

delivery performance of the mesoporous protein films than that

derived from the nature of the protein or protein networks.

4 Conclusions

In summary, a simple method was developed to prepare free-

standing mesoporous protein thin films by using ultrathin metal

hydroxide nanostrands as frames and pore templates through

a filtration process. Due to the mesoporous structures and the

multifunctional groups of the proteins, these films efficiently

demonstrated the transfer of negatively charged dye molecules

stimulated by pH. The delivery efficiency was as high as 0.335 g

g�1, which is about 3.7 times that of active carbon powders and

5.0 times that of metal hydroxide sludge. The form of the free-

standing thin film has further advantages, including no produc-

tion of secondary waste and not requiring further collecting

techniques. The biocompatibility of these protein films is

wonderful for drug delivery at physiological conditions. The

This journal is ª The Royal Society of Chemistry 2011

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advantage of these films for drug delivery is that the loading and

releasing process could be recycled. They are able to load

doxorubicin molecules at pH 6.6 and release them at pH 5, close

to the pH value of the tumour environment. These films show

promising applications for both recovering dyes from the dye

waste water, and for molecule drug delivery systems with

controllable releasing speed under the bio-environment. This

approach is also useful to synthesize other porous materials films

for various functional demands.

Acknowledgements

This work was supported in part by NSFC (21003105), the

Fundamental Research Funds for the Central Universities and

New Century Excellent Talents Program.

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