nanoparticle/polymer assembled microcapsules with ph...

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Nanoparticle/Polymer AssembledMicrocapsules with pH Sensing Property

Pan Zhang, Xiaoxue Song, Weijun Tong,* Changyou Gao

The dual-labeled microcapsules via nanoparticle/polymer assembly based on polyamine–saltaggregates can be fabricated for the ratiometric intracellular pH sensing. After deposition ofSiO2 nanoparticles on the poly(allylamine hydrochloride)/multivalent anionic salt aggregatesfollowed by silicic acid treatment, the generated microcapsules are stable in a wide pH range(3.0� 8.0). pH sensitive dye and pH insensitive dye are simultaneously labeled on the capsules,which enable the ratiometric pH sensing. Due to the rough and positively charged surface, themicrocapsules can be internalized by several kinds of cells naturally. Real-time measurementof intracellular pH in several living cells shows that the capsules are all located in acidic
organelles after being taken up. Furthermore, thenegatively charged DNA and dyes can be easilyencapsulated into the capsules via charge inter-action. The microcapsules with combination oflocalized pH sensing and drug loading abilitieshave many advantages, such as following thereal-time transportation and processing of thecarriers in cells.
P. Zhang, X. Song, Prof. W. Tong, Prof. C. GaoMOE Key Laboratory of Macromolecular Synthesis andFunctionalization, Department of Polymer Science andEngineering, Zhejiang University, Hangzhou 310027, ChinaE-mail: [email protected]

� 2014 WILEY-VCH Verlag GmbH & Co. KGaA, WeinheimMacromol. Biosci. 2014, 14, 1495–1504

wileyonlinelibrary.

1. Introduction

Microcapsuleshave longbeen recognized for theirpotential

biomedical applications in the fields of drug delivery,

confined biochemical reactions aswell as artificial cells.[1–3]

Among the various methods for fabrication of micro-

capsules,[1,4] the layer-by-layer (LbL) technique[5–7] has

gained tremendous attention in preparing engineered

multifunctional polymeric or hybrid capsules. Through

this method, capsules with well-controlled size and shape,

finely tuned capsule wall thickness and compositions, and

tailored functionalities have been successfully fabricat-

ed[8,9] and found great potential applications in biomedical

field.[10,11] Despite all the attractive advantages, one of the

drawbacks of the LbL technique is the time-consuming

multiple fabrication steps. Therefore, developing other

facilemethodswith great ease of fabrication and eventual-

ly scalable production of microcapsules is of both scientific

and technical importance. Recently, Wong and co-workers

have developed a newmethod of capsules formation based

on polyamine–salt aggregate assembly.[12] By simply

mixing of a cationic polyamine with a multivalent anionic

salt at ambient conditions, metastable polyamine–salt

aggregates (PSA) can be formed,[13,14] which can act as

templates for further addition of negatively charged shell

materials such as silica nanoparticles to form stable

capsules.[15] By controlling the fabrication conditions and

choosing different materials, capsules with different sizes,

DOI: 10.1002/mabi.201400259 1495com

Scheme 1. Schematic presentation of the preparation ofmicrocapsules via nanoparticles/polymer assembly based onpolyamine-salt aggregate followed by labeling with indicatordye (FITC) and reference dye (RBITC).

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P. Zhang, X. Song, W. Tong, C. Gao

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encapsulated cargoes as well as shell materials can be

obtained.[12] This kind of capsules also has shown promis-

ing application in photothermal therapy,[16] magnetic

resonance imaging[17] as well as protease-responsive

near-infrared imaging.[12] The easy fabrication process,

tremendous flexibility in building materials and tunable

size make the PSA capsules good candidates for drug

delivery applications.

As drug delivery vehicles, capsules can be taken up by

cells in many cases and experience an increasingly acidic

environment as they progress through the common

endocytic pathway.[18–20] The microenvironment of the

capsules in cells plays a significant role in the performance

of cargo’s therapeutic effect.[21] Eukaryotic cells are highly

compartmentalized and the unique function of individual

organelles depends on the establishment andmaintenance

of a narrow pH range,[22,23] thus capsules with pH sensing

property may report their location during cellular uptake.

Such knowledge is valuable for the design of better capsule

drug carriers.

Although many nano-sensors have shown their great

successes in the sensing and quantification of intracellular

ions in recent years,[24] microcapsules still have their own

advantages. Compared with nanosensors, microcapsules

allow the individual analysis of capsules via confocal

microscopy technique. Since vehicles internalized by cells

may experience different conditions as they entered cells at

different time point, individual analysis could give us a

moreprecise result of single vehicle. The LbLpolyelectrolyte

microcapsules with a pH sensing property have already

demonstrated this advantage. For example, Parak and co-

workers have fabricated LbLmicrocapsules loadedwithpH-

sensitivefluorophore SNARF-1asmobile local pHsensors to

monitor lysosomal pH changes upon stimulation.[18,25]

Donath and co-workers designed an LbLmultilayer system

by combining transporter and pH sensor for monitoring

intracellular degradation and expression of the loaded

plasmid DNA.[21] Uptake and processing of the particles

by cells are mainly investigated via flow cytometry,

and complemented by confocal laser scanning microscopy

(CLSM).

However, in most of above mentioned cases the

procedure to fabricate pH sensing capsules is quite

complicated which often first need the synthesis of dye-

conjugated polyelectrolytes. After multiple purification

process, the conjugated polyelectrolytes are used to form

capsules. The whole procedure is time-consuming and

may cause awaste of the dye-conjugatedpolyelectrolytes.

But in our study, this problem can be avoided since

the capsules are firstly fabricated and then the dye

labeling procedure is implemented, which is facile to

regulate the amount and ratio of the dyes labeled on the

capsules. Besides, the excess dyes can be easily removed

by centrifugation.

Macromol. Biosci. 201

� 2014 WILEY-VCH Verlag GmbH

In this work, we demonstrate that dual-labeled micro-

capsules via nanoparticles/polymer assembly based on the

PSA method can be fabricated for the ratiometric

intracellular pH sensing. Poly(allylamine hydrochloride)

(PAH) is ionically cross-linked with trisodium citrate

(Na3Cit) to formmetastable aggregates, and then negative-

ly charged SiO2 nanoparticles are deposited on these

aggregates to form stable capsules. To further stabilize

the capsules in awide pH range, silicic acid is added, which

can diffuse through the permeable wall and condense

within the shell and on the shell surface.[15] The generated

capsules are stable in a pH range of 3.0–8.0. Then pH-

sensitive dye (fluorescein isothiocyanate, FITC) and pH

insensitive dye (rhodamine B isothiocyanate, RBITC) are

covalently linked to PAH chains, which provides the

platform for ratiometric measurement (Scheme 1). The

structures of the capsules are characterized by CLSM,

scanningelectronmicroscopy (SEM)aswell as transmission

electronmicroscopy (TEM). The real timepHmeasurements

in living cells and colocatization study are performed using

CLSM. The loading of the capsules with negatively charged

cargoes is also demonstrated.

2. Experimental Section

2.1. Materials

Poly(allylamine hydrochloride) (PAH, Mw � 58kDa), fluorescein

isothiocyanate (FITC), rhodamine B isothiocyanate (RBITC), dimethyl

sulfoxide (DMSO), fluorescein sodium salt (Flu-Na), and 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphe-nyltetrazoliumbromide (MTT) were

purchased from Sigma-Aldrich. DNA was obtained from Sangon

BiotechCo., Ltd., Shanghai, China.Anaqueouscolloidal suspensionof

SiO2 nanoparticles (NPs) (20wt % silica, pH 3.5) was provided by

Jinan Yinfeng Silicon Products Co.,Ltd. These NPs have a diameter of

6–50nmbasedondynamic lightscatteringandazetapotentialvalue

of –15mVbyZetasizerNanoZS (Malver, UK). Tetraethyl orthosilicate

(TEOS) (>99%) was purchased from Aladdin Industrial Corporation.

Trisodium citrate (Na3Cit), citric acid monohydrate, and disodium

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Nanoparticle/Polymer Assembled Microcapsules . . .

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hydrogen phosphate dodecahydrate (Na2HPO4) were purchased

from Sinopharm Chemical Reagent Co.,Ltd. LysoTracker Green was

obtained from Invitrogen Co., USA. All chemicals were used as

received.Thewaterusedthroughoutall experimentswasprepared in

aMilliporeMilli-Q Referencepurification systemwith a resistanceof

18.2MV cm�1.

2.2. Fabrication of SiO2 NPs/PAH Capsules

PAH-citrate aggregateswerefirst prepared bymixing PAHsolution

(10mL, 2mgmL�1) and Na3Cit solution (25mL, 28.83mM) under

ultrasonication for 10s. The resulting suspension turned turbid

immediately, indicating the formation of PAH-citrate aggre-

gates.[14] After 2min aging, 25mL of 1.2wt% SiO2 NPs solution

(pH 3.5, adjusted with 1M HCl) was added into the prepared PAH-

citrate aggregates suspension under ultrasonication for 20 s. Then

themixed suspensionwasaged for 2hunder shaking to formstable

SiO2NPs/PAHcapsules. Toobtainmorestable capsules inawidepH

range, silicic acid treatment of the SiO2 NPs/PAH capsules was

further conducted. First, 0.31mL TEOS was added to a mixture of

24.44mL water and 0.25mL 0.1M HCl to form silicic acid clusters

with a composition of Si(OH)4.[13] After aged for 30min, the silicic

acid solution (25mL in total) was added into the above 60mL SiO2

NPs/PAH capsules suspension. After stirred for 30min at room

temperature, the capsules were separated by centrifugation

(4000 rpm, 3min), washed three times with water, and finally

dispersed in water for later use.

2.3. Fabrication of Dual-Labeled Microcapsules

The requireddyes (67mL FITCand185mLRBITC) fromstock solution

(1mgmL�1 in DMSO) were added into 52mL microcapsule

suspension (�1� 108 microcapsules per mL in NaHCO3/Na2CO3

buffer solution, pH 9.4). The mixture was agitated at room

temperature in dark for 12h. Then the labeled capsules were

separated by centrifugation (4000 rpm, 3min) and washed three

timeswithwater to remove free dyes. For the fabrication of RBITC-

labeled microcapsules, only RBITC was added and the following

procedures were the same as described above.

2.4. Cell Culture

Murine macrophage cell RAW 246.7, human HepG2 cells and

human endothelial cells (CRL-1730) were obtained from the Cell

Bank of Typical Culture Collection of the Chinese Academy of

Sciences (Shanghai, China). Primary human normal liver cells

(Hepli cells) were obtained from the First Affiliated Hospital,

College of Medicine, Zhejiang University. The RAW 246.7 cells and

HepG2 cells were cultured in Dulbecco’s modified eagle media

(DMEM, Gibco, USA) consisting of high-glucose, supplemented

with 10% fetal bovine serum (FBS, Sijiqing Inc., Hangzhou, China),

100UmL�1 penicillin, and 100mgmL�1 streptomycin and cultured

at 37 8C in a 5% CO2 humidified environment. The Hepli cells were

maintained in DMEM consisting of low-glucose, supplemented

with 10% FBS, 100UmL�1 penicillin, and 100mgmL�1 streptomy-

cin and cultured at 37 8C in a 5% CO2 humidified environment. The

CRL-1730 cells were maintained with regular growth medium


� 2014 WILEY-VCH Verlag Gmwww.MaterialsViews.com

consisting of high-glucose RPMI 1640 supplementedwith 10% FBS,

100UmL�1 penicillin, and 100mgmL�1 streptomycin and cultured

at 37 8C in a 5% CO2 humidified environment.

2.5. Generation of in Vitro Calibration Curve

In vitro pH calibration curves were obtained from CLSM images of

themicrocapsules inbufferswithartificial cytoplasm frompH3.8 to

7.8. To prepare buffers with artificial cytoplasm,[24] RAW 246.7 cells

(1� 106 per mL in water) were broken by sonication for 30min and

thenmixedwithbuffers (volumeratio1:2), and thepHwasadjusted.

Images were captured by a sequential line scanning model, with

excitation at 488nmand 543nm, respectively. The emission signals

were collected from 500 to 550nm for the green channel and from

570 to 630nm for the red channel, respectively. The background

fluorescence was zeroed by adjusting the gain and offset values. All

images were taken under the same settings (e.g., laser power, gain,

pinhole and zoom). Image processing and analysis were performed

on the Leica software (LAS AF Lite). Region of interest (ROI) was

selected based on peripheries of the microcapsules. The ratio of the

grey values between the two channels was calculated for

quantitative ratiometric analysis. 30 individual capsules in three

images were analyzed for each pH value measurement. Calibration

curve was generated by plotting the average values together with

the standard deviation versus pH values.

2.6. CLSM Imaging and Localized Intracellular pH

Measurement

The RAW 246.7 cells were seeded in a 20-mm glass bottom cell

culture dish at a density of 7� 104 cells per well at 37 8C. After24h, the culture medium was changed and dual-labeled micro-

capsules were added at a capsule/cell ratio of 50:1. The concentra-

tion of the capsules stock solution was determined by a counting

chamber. Cells were incubated with microcapsules for 24h (and

3h and 6h for the kinetic study). Before CLSM imaging, the cells

were rinsed 3 times with phosphate-buffered saline (PBS) to

remove non-adhered microcapsules and then kept in culture

medium without phenol-red for CLSM imaging. The cells were

mounted on the microscope stage equipped with a temperature

controlled incubator box and CO2 supply for optimal growth

conditions during observation. Cells were imaged in a sequential

line scanning mode by 488 and 543nm lasers along with a bright

field image. Generally, 20–40 positions were selected and more

than 100 capsules were analyzed. The same procedures were

done for the intracellular pH measurement of HepG2, CRL-1730

and Hepli cells after 24 h uptake of microcapsule sensors.

2.7. Co-localization

RAW 246.7 cells were seeded in a 20-mm glass bottom cell culture

dish at a density of 7�104 cells per well at 37 8C for 24h. After

treated with RBITC-labeled microcapsules at a concentration of 50

capsules per cell for 24h, the cells were washed 3 times with PBS

and incubatedwith LysoTracker Green at 37 8C for another 30min.

They were then washed and imaged by CLSM as described above.

Images processing were conducted using Image J (NIH).

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2.8. Loading of Negatively Charged Molecules

Due to the remaining positive charges of PAH, negatively charged

molecules can be easily loaded into the capsules. Fish sperm DNA

was chosen as the negatively charged biomacromolecular model

drug. The unlabeled SiO2 NPs/PAH microcapsules (�1.25� 109

microcapsulespermL)were incubated inDNAsolution (1mgmL�1,

0.05MNaCl) for12hunderconstantshaking.The resultantcapsules

were washed with fresh 0.05M NaCl solution thrice and finally

washed twicewithwater. The supernatantwas carefully collected

for UV–vis spectroscopy measurement (Shimadzu UV-vis 2550).

The concentration of the supernatant was determined at 260nm

and by referring to a calibration curve. The amount of loaded DNA

was calculated by the difference of the feeding drug to the

determined in the supernatant. All data were averaged from 3

parallel experiments. For fluorescent microscope (Zeiss Axiovert

200) observation, FITC-DNA was synthesized according to the

literature.[26,27] Low-molecular-weight model drug Flu-Na was

loaded in the same way.

2.9. Characterizations

2.9.1. Confocal Laser Scanning Microscopy (CLSM)

Confocal images were taken with Leica TCS SP5 confocal scanning

system equipped with a 63�oil immersion objective. A drop of

capsules suspensionwasappliedontoa glass slide for visualization

after theywereprecipitated.Todeterminethecapsulesdiameter,at

least 200 capsules, dispersed in PBS, were analyzed by Image J

software.

2.9.2. Scanning Electron Microscopy (SEM)

Adropof SiO2NPs/PAHcapsules suspensionwas applied on a glass

slide and dried in air overnight. After sputtered with gold, the

samples were observed with HITACHI S-4800 instrument at an

operation voltage of 3 kV.

2.9.3. Transmission Electron Microscopy (TEM)

The SiO2 NPs/PAH capsules were washed with graded ethanol/

water solutions. The sample was embedded into epoxy resin and

ultramicrotomed into thin sections, whichwere transferred onto a

carbon film-coated copper grid and observed by a Philips Tecnal-10

TEM.

2.9.4. Zeta potential measurements

ZetapotentialmeasurementswereperformedonZetasizerNanoZS

(Malver, UK). The results were averaged from 3 parallel

measurements.

2.9.5. Fluorescence Spectroscopy

For the pH-sensingmeasurement, universal buffer solutions of pH

ranging from 3.8 to 7.8 were first prepared using stock solutions of

Na2HPO4 (250mL,0.2M) andcitric acidmonohydrate (250mL,0.1M)

with different volumes.[28] Then the series of buffer solutionswere

mixed with water (volume ratio of 2:1). The pH was measured by



FE-20-FiveEasyTM pH meter (Mettler Toledo, Switzerland). Then

microcapsules were mixed with buffer solutions (�1� 108 micro-

capsules permL). The emission signals in the ranges of 510–560nm

(excited at 488nm) and 570–620nm (excited at 543nm) were

collected using FL-55 fluorometer (Perkin Elmer, Japan),

respectively.

2.9.6. Fluorescence reversibility of microcapsules with pH

ThepHof themicrocapsules buffer solutionwas switchedbetween

4.0 and 7.0 repeatedly by 2M HCl or NaOH. The fluorescence

emission spectra excited at 488nmand 543nmwere recorded. The

ratio of signal (R¼ I525/I585) was calculated from the fluorescence

intensity at 525nm and 585nm.

3. Results and Discussion

3.1. Fabrication of SiO2 NPs/PAH Microcapsules with

pH Sensing Property

Compared with other techniques, the polyamine–salt

aggregates assembly method has several advantages. The

fabrication process only involves simplemixing and can be

conducted at mild conditions in aqueous solution. By

changing the fabrication conditionsandbuildingmaterials,

the size as well as the composition can be tuned to fulfill

various applications. Furthermore, different cargoes can be

easily encapsulated by using them as one of building

materials.[12] Thus this kind of capsules can be easily

fabricated in large scale and are good candidates for

functional drug delivery vehicles.

In our study, the capsules were fabricated by this

technique. The amine groups on PAH chains can be

ionically cross-linked by trivalent citrate anions to form

aggregates immediately when mixed with Na3Cit. Then

negatively charged silica nanoparticles were assembled on

these aggregates, which served as templates to generate

SiO2NPs/PAHmicrocapsules.[13] But thegeneratedcapsules

were not stable below pH 4, which is near the isoelectric

point of silica.[29] In this case the capsules were disas-

sembled due to the charge lose of silica. To further stabilize

the capsules, the silicic acid treatment was applied. The

generated capsules can hold stable structures in buffers

with pH ranging from 3.0 to 8.0, and thereby meet the

intracellular pH sensing requirement. The obtained capsu-

les can be well dispersed in aqueous solution (Figure 1a).

They have an average size of 1.7� 0.3mm. The capsules are

positively charged with a zeta potential of 20.9� 0.7mV

due to the presence of PAH amine groups exposed at the

capsule surface. This size distribution as well as the

positively charged surface can facilitate cell uptake of

the capsules. Bymixingwith dye solutions, FITC and RBITC

were covalently linked to the microcapsules through

the reaction between their isothiocyanate groups and the

amino groups of PAH. The capsules showahollowstructure

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Figure 1. CLSM images of the dual-labeled microcapsule dispersed in pH 7.0 buffer solutions: a) FITC channel and b) RBITC channel. c) Lineintensity profiles along the white line shown in the inset picture.


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with bright green and red fluorescence (Figure 1a,b).

According toWong’s results,[15] the silicic acid nanoclusters

have some capacity for disassembling the PAH-citrate core,

and the salt solutions used in the fabrication and labeling

steps also can screen the charge interaction between PAH

and citrate, resulting the disassembly of the core. However,

the electrostatic interaction between PAH and silica NPs is

stronger than that between PAH and citrate because the

NPs have more negative binding sites than citrate anions,

thus the shell is stable during the fabrication and labeling

process. The released PAH can diffuse out and finally a

hollow capsule can be obtained. After labeled with

fluorescent dyes, the fluorescence of labeled PAH can

indicate their distribution. According to the line intensity

profile along the line across the capsule in Figure 1c,most of

the PAH molecules are located in the shell. However there

are still small amount of them in the interior. This result is

consistent with previous report.[13]

The capsules aremechanically robust and can keep their

shape even after drying (Figure 2a). The corresponding

magnified image reveals that the surface of capsule is very

Figure 2. a) SEM image and b) TEM image of the dual-labeledmicrocapThe inset of (b) is a part of a magnified microcapsule shell; scale ba



rough and is composed of many silica nanoparticles.

According to a recent report,[30] the silica nanoparticles

assembled on vehicles surface can increase nanoscale

surface roughness which mimics viral surface topography.

This surface can enhance both binding towards biomole-

cules and cellular uptake efficacy. Thus the improvement of

the cellular delivery performance can be expected. The

hollow structure as well as more detailed structure

information of shell was further revealed by the ultra-

microtomed sections (Figure 2b). The shell of the micro-

capsules has two distinct parts as shown in the TEM image

(Figure 2b), i.e. the outer-part is mainly composed of silica

NPs and PAH, and the inner-part of the shell is mainly

composed of PAH and silicic acid species. The silicic acid

oligomeric species can diffuse through the permeable silica

NPs/PAH shell and subsequently deposit within the wall

and on the inner shell wall surface.[15,31] The microcapsule

structure plays a pivotal role in protecting the sensor dye

and reference dye from intracellular interferences as

reported earlier,[32] meanwhile guarantees a fast response

to the outside changes in pH.

sules. The inset of (a) is a magnifiedmicrocapsules; scale bar, 0.5mm.r, 100nm.

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Figure 4. In vitro calibration curves of the capsule sensorsgenerated from CLSM images. Calibration was done in artificialcytoplasm.

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1500

3.2. pH-Sensitivity and Reversibility of the

Microcapsule Sensors

The sensing property of the microcapsules was tested for

their pH sensitivity at various pH values via fluorescence

spectroscopy. FITC and RBITC were excited at 488nm and

543nm, respectively. Fluorescence spectra of the micro-

capsules at different pHs were plotted in Figure 3a. The

emission peak at approximately 525nm is assigned to FITC,

which is enhanced when pH is increased from 4.0 to 7.8. In

contrast, the emission intensity at 585nm of RBITC is

relatively stable. This is very suitable for ratiometric pH

measurement, which allows for compensation of inhomo-

geneities of the fluorophores loaded in capsules, size

distribution of capsules,[33] light source fluctuation and

photobleaching.[34] The reversibility and reproducibility of

themicrocapsule sensorsarealsovery important forprecise

intracellular pH measurement. When the pH was repeat-

edly switched between 4 and 7, the ratio of FITC/RBITC

fluorescence emission signals (R¼ I525/I585) showed good

reversibility and reproducibility in three cycles (Figure 3b).

3.3. Intracellular pH Sensing

For intracellular pH sensing, a ratiometric pH calibration

curvewas first generated via CLSM images, as illustrated in

Figure 4. To mimic the intracellular environment, artificial

cytoplasm was used to construct the calibration curve.[24]

The calibration curve was then fitted to a sigmoidal

function[28] and the specific pKa value was calculated as

being 6.06. The value is slightly small than the reported pKa

value of FITC (6.4),[35] which may due to the conjugation of

dyes onto PAH molecules.

Intracellular pH measurement within RAW 246.7 cells

was then conducted by confocalmicroscopy. After incubat-

Figure 3. a) Fluorescent spectra of microcapsules in buffer solutionsbetween pH 4 and 7.



ing cells in the presence of dual-labeled microcapsule for

3 h, 6 hand24h, the cellswerewashed three timeswithPBS

and then imaged by CLSM (Figure 5). The microcapsule

sensors localized in different pH environments show

different colors. The capsules in neutral and alkaline

environment show green color, whereas those in orange/

yellow color are highly possible to enter cells and are

localized in acidic environment. Therefore, according to the

color of the microcapsules, the intracellular capsules are

easily distinguished from those adhered to the outer cell

membrance or in the medium. This performance is in

accordance with the previous report.[36] Through single

capsule analysis, the precise pH value distribution of

several images containing more than 100 capsules at each

at various pH values and b) pH reversibility study of microcapsules

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Figure 5. CLSM images of RAW 246.7 cells with internalized microcapsules after a) 3 h, b) 6 h, and c) 24 h. Left column: bright filed images;middle column: overlay images of the green and red channels showing the microcapsules; right column: overlay images of the bright filedimages and the corresponding fluorescence images. Scale bar, 10mm.


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timepointwascalculatedaccording to thecalibrationcurve

(Figure 6). After 3 h, the mean pH value of intracellular

microcapsules is 5.1� 0.5 (mean� SD, n¼ 184). Approxi-

mately 72% of the capsules are in the pH range of 4.0–5.5

whilenearly28%are in thepHrangeof5.5–6.5 (Figure6). By

referring to the reported endocytic pathway (early

endosomes, pH 6.5; late endosomes and lysosomes, pH

4.0–5.5),[22,37] it is speculated that many capsules have

reached the late endosomes and lysosomes while a small

part are locatedwithin the early endosomes.After 6h,more

capsules are taken up with an average pH of 5.0� 0.4

(mean� SD, n¼ 135). 91% of the capsules are in the pH

rangeof4.0–5.5, indicating thatmoreandmorecapsulesare

transported from the early endosomes into themore acidic

late endosomes and lysosomes (Figure 6). After 24h,

the mean pH value is 5.0� 0.4 (mean� SD, n¼ 188), which

is same to that of 6h, indicating that the microcapsuls

are finally entraped in the acidic enviroment. In contrast,

the extracellular pHdeterminedby extracellular capsules is

rather high with an average value of 6.9� 0.5 (mean� SD,

n¼ 9).



The above results are also in linewith the co-localization

study. The RAW 246.7 cells were treated with the RBITC-

labeled microcapsules for 24h. Then the cells were stained

with LysoTracker Green to label the acidic organelles. The

green and red channels are mostly overlapped, showing

that the capsules were surrounded by acidic vesicles

(Figure 7). The pathway andmechanisms of internalization

of capsules have been investigated by many research-

ers.[18,19,21,38] These findings also suggest that the internal-

ized microcapsules are transported to the acidic

compartments.

The SiO2 NPs/PAH capsuleswere further incubatedwith

other three kinds of cells to check the cell type-dependent

delivery performance. HepG2 cells, Hepli cells and CRL-

1730 cells were chosen for the study. HepG2 cell line is

human hepatoma cells, Hepli cell line is primary human

normal liver cells, and CRL-1730 cell line is human

endothelial cells. After being incubated with the micro-

capsules for 24 h, the three kinds of cells were washed and

observed via CLSM (Figure 8). The microcapsules all show

good cellular uptake performance regardless of the cell

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Figure 6. pH distribution of the microcapsule sensors at different incubation time which were generated from CLSM image, correspondingto Figure 5.

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types. The silica nanoparticles assembled on capsule

surface can increase the surface roughness to mimic viral

surface topography, thus furthermore promote the cellular

uptake as recognized previously.[30] The positively charged

surface of capsules also can facilitate cellular uptake.[39]

Themicroenvironmentof thecapsulesafter enteredHepG2

cells suffered a mean pH value of 4.6� 0.6 (mean� SD,

n¼ 241) and in Hepli cells had a mean pH of 4.5� 0.5

(mean� SD, n¼ 106). Similarly, after internalized by CRL-

1730 cells, the capsules were located in an environment

with an average pHof 4.5� 0.5 (mean� SD,n¼ 145). These

results indicate that SiO2NPs/PAHcapsules canbeuptaken

by different types of cells, and after internalized, they are

all located in acidic compartments irrespective of the

cell types.

3.4. Loading of Negatively Charged Molecules into

Capsules

Since the capsules have positively charged surface, and PAH

chains are dispersed both in the shell and the interior,

negatively charged molecules can be easily loaded through



the charge interaction.[39–41] DNA was chosen as the

negatively charged biomacromolecular model drug. The

strong green fluorescence emission from the capsules

indicates the successful loading of FITC-DNA in the micro-

capsules (Figure 9). Quantitative measurement revealed

that0.26pgDNAwas loaded inonecapsule. Low-molecular-

weight model drug sodium fluorescein was loaded as well,

and 0.38pg sodium fluorescein was entrapped in one

capsule. These results demonstrate that the capsules have

the potential as drug delivery vehicles, encapsulating

negatively charged small molecular drugs or biomacromo-

lecules such as pDNA and siRNA for gene therapy.

4. Conclusion

In summary, stable nanoparticles/polymer assembled

microcapsules were fabricated via the polyamine-salt

aggregatesassemblymethod, followedbythenanoparticles

deposition and further silicic acid treatment. After being

labeled with fluorescein and rhodamine B, which act as

indicator dye and reference dye respectively, the capsules

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Figure 7. Co-localization of the microcapsules. a) LysoTracker Green labeled the endo/lysosomes of RAW 246.7 cells. b) RBITC labeledmicrocapsules. c) Overlay image of the red and green channel. d) Overlay of the two fluorescence channels and the bright field image. Scalebar, 10mm.


www.mbs-journal.de

were endowedwithpHsensingproperty. Theywereused to

assay the intracellular pH via ratiometric measurement.

Due to the rough and positively charged surface, the

microcapsules were internalized by several kinds of cells

naturally, thus indicating the good cellular delivery perfor-

Figure 8. CLSM images of microcapsules internalized by a) HepG210mm.



mance. After internalized, the capsules were located in

acidic organelles. The microcapsules showed a good

performance in monitoring the intracellular local pH in

real-timemanner. Additionally, the capsules canact as drug

delivery vehicles by successfully encapsulating negatively

cells, b) Hepli cells, and c) endothelial cells after 24 h. Scale bar,

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Figure 9. Fluorescent image of FITC-DNA encapsulated in themicrocapsules after incubation for 12 h. Scale bar, 10mm.

www.mbs-journal.de


1504

charged drugs. The nanoparticles/polyamine assembled

microcapsules with combined pH sensing property and

ability for drug loading are very useful for the real-time

monitoringof intracellular localpHduring thedrugdelivery

process.

Acknowledgements: This study is financially supported by theNatural Science Foundation of China (Nos. 21174130, 21374101 and51120135001) and theMarie Curie project (PIRSES-GA-2013-612673).

Received: May 26, 2014; Revised: June 18, 2014; Published online:August 1, 2014; DOI: 10.1002/mabi.201400259

Keywords: cellular uptake; loading; microcapsules; nanoparticle/polymer assembly; pH sensing

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