Journal of Microencapsulation, 2012; 29(5): 437–444� 2012 Informa UK Ltd.ISSN 0265-2048 print/ISSN 1464-5246 onlineDOI: 10.3109/02652048.2012.655333

Hematoporphyrin encapsulated PLGA microbubble for contrastenhanced ultrasound imaging and sonodynamic therapy

Yuanyi Zheng1, Yaping Zhang1, Meng Ao1, Ping Zhang1, Hui Zhang1, Pan Li1, Lang Qing2,Zhigang Wang2 and Haitao Ran1

1Second Affiliated Hospital of Chongqing Medical University, Chongqing 400010, China and 2Institute of UltrasoundImaging of Chongqing Medical University, Chongqing 400010, China

AbstractThe purpose of this study was to develop a sonosensitizer-loaded multi-functional ultrasound (US) con-trast agent for both tumour therapy and imaging. The hematoporphyrin (HP)-encapsulated poly(lactic-co-glycolic acid) microbubbles (HP-PLGA-MBs) were prepared and filled with perfluorocarbon gases. Theenhancement of US imaging and its sonodynamically induced anti-tumour effect were evaluated by bothin vitro and in vivo experiments. The HP-PLGA-MBs have a narrow size distribution and smooth surface witha mean diameter of 702.6� 56.8 nm and HP encapsulation efficiency of 63.50� 1.26% and drug-loadingefficiency of 2.15� 0.13%. The HP-PLGA-MBs could well enhance the ultrasound imaging both in vitro andin vivo. A significant anti-tumour effect was obtained by HP-PLGA-MBs mediated sonodynamic therapy. Thetumour growth rate and the tumour proliferation index were the lowest in the HP-PLGA-MBs plus sonica-tion group. And the tumour cell apoptotic index was the biggest in the HP-PLGA-MBs plus sonicationgroup. In conclusion, a sonosensitizer-loaded multi-functional contrast agent was constructed and thefeasibility was demonstrated, which might provide a novel strategy for tumour imaging and therapy.

Keywords: hematoporphyrin, PLGA, ultrasound contrast agent, sonodynamic therapy, tumor

Introduction

Sonodynamic therapy (SDT) is a promising approach to the

treatment of tumours, which has been clinically applied to

cancer, such as breast cancer (Wang et al., 2009; Barati and

Mokhtari-Dizaji, 2010). The sonosensitizers, mainly hema-

toporphyrin (HP) or hematoporphyrin derivatives (HPDs),

are accumulated in tumour tissues and excited by exposure

to the appropriate frequency and energy of ultrasound,

resulting in tumour cell killing by activated oxygen pro-

duced by the sonosensitizers (Kuroki et al., 2007). While

like other chemotherapy drugs, sonosensitizers have signif-

icant side effects, such as blister formation, skin hyperpig-

mentation, ocular discomfort, pruritus, pain at injection

site and urticaria (Wooten et al., 1988).

The rapid development of high molecular chemistry and

nanomedicine holds promise for solving the problems

of current chemotherapy, such as side effects of

chemotherapeutic drugs (Rapoport et al., 2009). Various

high molecular drug delivery modalities have been sug-

gested for decreasing the side effects caused by chemother-

apeutic drugs, which are commonly based on drug

encapsulation in nano or microparticles, etc. The encapsu-

lation may dramatically increases drug concentration in an

aqueous environment and offers new life to bioactive com-

pounds previously abandoned due to low aqueous solubil-

ity (Rapoport et al., 2009). And in order to further improve

the outcome of tumour therapy, the stimuli-responsive

drug delivery modalities have been developed. The pay-

load will be released in response to environmental or phys-

ical stimuli, such as pH, hyperthermia, light, or ultrasound,

which had important implications for tumour therapy.

Among the physical stimuli, ultrasound irradiation has

been proved to be a good tool for the controlled drug

release study. It has been demonstrated that ultrasonic

irradiation could efficiently trigger drug release from the

Address for correspondence: Haitao Ran, Second Affiliated Hospital of Chongqing Medical University, 76 Linjiang Road, Chongqing 400010, China.Tel: 86-23-63693709. Fax: 86-23-63809074. E-mail: rht66@163.com

(Received 22 Aug 2011; accepted 19 Dec 2011)http://www.informahealthcare.com/mnc

437

drug carriers and could transiently alter cell membrane

permeability, resulting in effective anti-tumor effect

(Watanabe et al., 2008; Xing et al., 2008).

The encapsulation of sonosensitizers inside the high

molecular particles might have several benefits. First, the

encapsulation should be able to alleviate the side effects of

sonosensitizers. Second, the application of ultrasound

could not only activate the sonosensitizers but also locally

(also called passive targeting (Liu et al., 2010)) release the

sonosensitizers, which was considered be able to improve

the efficiency of chemotherapy and further alleviate the

side effects. Third, the high molecular particles could be

fabricated as a drug carrier and an ultrasound contrast

agent at the same time. The combination of the contrast

enhanced diagnostic ultrasound imaging and ultrasound

mediated therapy could be especially attractive for reasons

of simplicity and cost-effectiveness.

Recently, the term of theranostics has become a hot

topic in both chemistry and medicine research field,

among which the drug-loaded ultrasound microbubbles

were explored (Ke et al., 2011). Ultrasound contrast

agents (UCAs) are microbubbles composed of a thin albu-

min, galactose, lipid, or polymers, Shell filled with a heavy

gas, such as perfluorocarbon or nitrogen. UCAs have a high

degree of echogenicity, which is the ability of an object to

reflect the ultrasound waves. The echogenicity difference

between the gas in the microbubbles and the soft tissue

surroundings of the body is immense. Thus, ultrasonic

imaging using UCAs enhances the ultrasound backscatter,

or reflection of the ultrasound waves, to produce a unique

sonogram with increased contrast due to the high echo-

genicity difference. With the use of UCAs, the resolution

and sensitivity of clinical ultrasound imaging have been

greatly improved. Nowdays, UCAs have been widely used

in clinical practice in Europe and China as ultrasound con-

trast agents for tumour imaging. And UCAs also have been

widely reported as effective drug carriers and enhancers of

drug and gene delivery in experimental studies (Ren et al.,

2009). The use of ultrasound microbubbles as drug carriers

is very attractive. It would combine cost-effective ultra-

sound imaging with ultrasound-mediated therapy, and

ultrasound is the only clinical used imaging modality

for real time guidance and monitoring of radiofrequency

therapy of tumours (Solbiati et al., 2004). However,

these commonly used microbubbles are not a good drug

carrier because their drug-loading efficiency is low and

they are made from either lipid or albumin, such as

Sonovue or Optison, which have very short circulation

time (minutes).

The way to solve the side effects problem of sonosensi-

tizers and to develop an effective theranostic at the same

time may consist in developing drug-loaded high molecu-

lar ultrasound microbubbles, such as poly(lactic-

co-glycolic acid) (PLGA) or chistan microbubble, etc.

Their important properties combine drug carrying and

enhancing the ultrasound contrast of tumours. While to

best of our knowledge, high molecular microbubbles car-

rying sonosensitizers for both ultrasound imaging and

sonodynamic therapy have never been reported yet.

Here, we describe therapeutic and ultrasound-imaging

properties in these high molecular microbubbles that were

explored using the mouse subcutaneous tumour model.

Materials and methods

Fabrication of hematoporphyrin (HP) encapsulated

poly(lactic-co-glycolic acid) (PLGA) Microbubbles

(HP-PLGA-MBs)

HP-PLGA-MBs were fabricated using a modified double

emulsion (water/oil/water) method followed by solvent

evaporation (Zhou et al., 2008; Ao et al., 2010). Briefly,

100 mg of PLGA (75:25; Sigma) was dissolved in 2 mL meth-

ylene chloride, and 200mL of 10 mg/mL Hematoporphyrin

(HP) in Milli-Q water was added to the organic solution

and subjected to strong vibration for 45 s using an ultra-

sound microbubble vibrator (Chonqging Anbijie Inc,

China), to create the first emulsion (W/O). The resulting

(W/O) emulsion was then poured into cold (4�C), 5% poly-

vinyl alcohol solution and homogenized using homoge-

nizer (FJ-2000,Shanghai sample model Inc, China) for

5 min at 9000 rpm, to create the second emulsion

((W/O)/W). The double emulsion was then poured into a

2% isopropanol solution and stirred at room temperature

for 1 h. The capsules were then collected by centrifugation

and washed three times with deionised water. The capsules

were mixed with 3 mL deionised water and frozen in a

�80�C freezer, lyophilized for 48 h. The dried samples

were filled with perFuorocarbon gases and stored in a free-

zer at �20�C until used.

Characterization of HP-PLGA-MBs

Particle size analysis

Particle size, size distribution and concentration were

determined using a Malvern Zetasizer Nano ZS (Malvern

Instrument, UK) and a coulter counter (Multisizer III;

Beckman Coulter, USA). BrieFy, 1 mg/mL of HP-PLGA-

MBs solution was prepared in double distilled water. Size

measurements were performed in triplicates following a

1/100 (v/v) dilution of the HP-PLGA-MBs suspension in

MilliQ water at 25�C.

Scanning electron microscope (SEM) studies

The surface morphology of microbubbles was character-

ized by using a scanning electron microscope (KYKY2000).

Assessing the encapsulation efficiency

and loading efficiency

Drug encapsulation efficiency in the HP-PLGA-MB micro-

bubbles was assessed by high-performance liquid

438 Y. Zheng et al.

chromatography. The concentration of hematoporphyrin

in the solution was obtained from the standard curve of

hematoporphyrin in dimethyl sulfoxide (DMSO), which

relates absorbance and concentrations. Briefly, 4 mg of

freeze-dried HP-PLGA-MB microbubbles were dissolved

in 2 mL DMSO in a tube, and 4 mg of dry blank PLGA

microbubbles were taken as the control group. HP content

in the solution was determined by high-performance liquid

chromatography. The encapsulation efficiency of HP in

microbubbles was calculated using the following

equation (Ran et al., 2005). Drug encapsulation efficiency

(EE):

EE½%� ¼ ðW1=WTHPÞ � 100%;

where: W1 is the total drug amount in the HP-PLGA-MB

microbubbles; WTHP is the total weight of HP used in the

preparation of the HP-PLGA-MB microbubbles. Loading

efficiency (LE):

LE½%� ¼ ðW1=WTMBÞ � 100%;

where: W1 is the total drug amount in the HP-PLGA-MB

microbubbles; WTMB is the total weight of HP-PLGA-MB

microbubbles.

In vitro drug release experiment

In vitro sonication experiment was performed in order to

assess the drug release behaviour in the sound field, and

hence its suitability as a platform for triggered drug delivery

(Janoria and Mitra, 2007). The parameters of the sonication

was the similar to our custom-used parameter for cell gene

transfection in vitro (Ren et al., 2009). Sample measuring

10 mg was added to 50 mL of 37�C PBS within the sample

holder. The agent was then insonated using the 1 MHz

transducer at 2 W/cm2 (Model UTG 1025, Institute of

Ultrasound Imaging of Chongqing Medical Sciences,

Chongqing, China). The release medium was withdrawn

at predetermined time intervals, and replaced with fresh

soaking medium (1 mL) each time. HP content in the aque-

ous solution was analyzed using the high-performance

liquid chromatography method. Controls were performed

using the same setup and time scale, but without

sonication.

In vitro ultrasound imaging experiment

Imaging phantom overview

Figure 1 shows the experimental setup consisting of poly-

ethylene tubing (internal diameter (ID): 1 mm. Western

Analytical Co., CA) embedded into a 3% gelatine phantom

in a 20 cm� 10 cm� 5 cm tank, a pump and a rack which

was used to hold the ultrasound probe on the gel phantom.

Flow was generated by the pump (Watson-Marlow Sci400,

Watson-Marlow Alitea, England). The signal produced by

the microbubbles at the concentration of 108/mL was cap-

tured by the harmonic ultrasound probe.

Ultrasound image acquisition

Contrast-enhanced harmonic images were obtained using

a 5–12 MHz linear-array ultrasound transducer (IU22;

Philips Medical Systems, France). The default harmonic

imaging mode (mechanic index (MI): 0.08, frame rate

(FR): 45 fps, depth: 3.5 cm) was used for the imaging. The

microbubbles were administered with the probe fixed by

means of a custom-designed clamp to maintain position

above the tube. The image intensity (gray value) of region

of interest (ROI) was measured using an ultrasound image

analysis software (Model: DFY, Chongqing Medical

University, China).

In vivo ultrasound imaging and therapy of mouse tumour

Cell culture and subcutaneous tumour model

(Zhang et al., 2010)

The mouse H22 hepatocellular carcinoma cells (H22 cells)

were maintained at 37�C with 5% CO2 in completed RPMI

supplemented with 100 mg/mL penicillin, 100 mg/mL

streptomycin, and 10% fetal bovine serum. Thirty-

six-week-old C57BL/6J mice were bred in house and were

kept under standard housing conditions. For tumour injec-

tion, cells were harvested by trypsinization, centrifuged,

and resuspended at a density of 2� 106 cells/mL.

Tumours were inoculated subcutaneously on the left

flank with a 20 mL of cell mixture by using a 25-gauge

needle. Tumours were monitored weekly for growth prog-

ress via ultrasound. The tumours were used for the exper-

iment once they reached a diameter of approximately

0.5 cm. All experiments were conducted under protocols

approved by the Institutional Animal Care and Use

Committee at Chongqing Medical University.

In vivo ultrasound imaging and ultrasonic exposure

Thirty mice were randomized into five groups (six rats each

group) for therapy experiment. All groups were subjected

to ultrasonic exposure except the first group. The first con-

trol animal group (Saline group) was taken as a blank con-

trol by the injection of saline only. The second group

(Saline-US group) was injected the saline and subjected

to ultrasonic exposure. The third group (PLGA-MBs-US

Figure 1. Imaging phantom overview.

Hematoporphyrin encapsulated PLGA microbubble 439

group) was injected the bubbles without hematoporphyrin

inclusion and subjected to ultrasonic exposure. The fourth

group (HP-US group) was injected hematoporphyrin

(20 mg/kg) and subjected to ultrasonic exposure. The fifth

group (HP-PLGA-MBs-US group) was injected hematopor-

phyrin inclusion (20 mg/kg) bubbles and subjected to

ultrasonic exposure. The mice were anesthetized with iso-

flurane (1.5% vol. at 2 L/min) via a nose cone. Body tem-

perature was maintained at 37�C.

The same ultrasound image acquisition method as in

the in vitro experiment was employed for the in vivo exper-

iment except the probe was fixed on the tumours instead of

the gel phantom. The ultrasound microbubbles had a final

concentration HP at 20 mg/kg. For each single injection, a

syringe with 23 gauge needles was used to draw the bubble

solution according to each mouse’s body weight. The sus-

pension was then injected through tail vein of mice.

Immediately after administration, the injection port was

flushed with 0.3 mL of saline solution. The procedure of

the enhancement of ultrasound imaging was recorded as

DICOM clips. And when the microbubbles arrive at the

tumour observed by the ultrasound imaging, the ultra-

sound imaging probe was moved away and ultrasonic

probe (frequency: 1 MHz, intensity: 2 W/cm2) was transder-

mally applied on the tumours and continuously sonicated

for 2 min. The imaging and therapy procedures were

repeated every other day for a total three times.

Evaluation of anti-tumour effect

The curative effect was evaluated by measuring the tumour

growth and comparing it to the control groups after treat-

ment. The long (a) and short (b) diameters of the tumours

and the weight of mice were measured every day after

treatment. Tumour size was calculated as: volume¼

�/6[(aþ b)/2]3 and the volume inhibition ratios were cal-

culated as: (1-average tumour volume of treated group/

average tumour volume of the control group)� 100%.

Fifteen days after the treatment, the mice were killed and

the tumours were dissected out and weighted. The tumour

inhibition ratios of the tumour weight were calculated as:

(1-average tumour weight of treated group/average tumour

weight of the control group)� 100%.

Histopathologic examination

Tumours were harvested on the fifteenth day of treatment.

The tissue was fixed in 10% formalin solution and embed-

ded in paraffin. Slides of 5mm thickness were cut and

stained with hematoxylin and eosin (H&E). The TdT-

mediated dUTP-biotin nick-end labelling (TUNEL) assay

was performed to evaluate the apoptotic cell death. The

TUNEL-positive cells were counted in 10 randomly

selected Eelds using a light microscope at 200�magniEcat-

ion. The apoptotic index (AI) of a tumour was deEned as

the number of apoptotic cells in 100 tumour cells. The pro-

liferative cell nuclear antigen (PCNA) was measured by

immunohistochemical staining method using a mouse

monoclonal antibody to PCNA (dilution 1:50) (Cormedica

Corp., CA, USA). The PCNA-positive cells were counted in

10 randomly selected Eelds using a light microscope at

200� magniEcation. The Proliferating index (PI) of a

tumour was deEned as the number of PCNA positive cell

nuclei in 100 tumour cells. All histological processing was

carried out by the Histology Core facility of the Chongqing

Medical University. Histopathological examination of the

tumours was conducted by blinded analysis.

Statistical analysis

All data were expressed as means� SD. The differences

among the groups were analyzed of variance (one-way

ANOVA), the difference of intensity between the two

groups in the in vitro ultrasound imaging study was ana-

lyzed of t-test, p5 0.05 was considered to be significant

and p5 0.01 was considered to be extremely significant.

Results and discussion

Characterization of morphology of the

hematoporphyrin-inclusion HP-PLGA-MBs

In this study, we prepared the PLGA microbubbles carrying

hematoporphyrin (HP-PLGA-MBs) as an imaging agent as

well as a controlled released sonodynamic therapeutic

agent. We used SEM to characterize the morphological

properties of these particles. Figure 2 shows the SEM

image of HP-PLGA-MBs. The SEM analysis showed that

the PLGA particles were spherical and had a smooth sur-

face. The SEM image reveals HP-PLGA-MBs to be com-

prised of essentially spherical microparticles.

The mean diameter of the PLGA microbubbles was

702.6� 56.8 nm and the mean concentration was 108

microbubbles/mL. The particle size of HP-PLGA-MBs is

important for both safety and efficacy. Most of the micro-

particles must be smaller than the size of a red blood cell

(8 mm). The particle size data demonstrate that the most of

the HP-PLGA-MBs were sub-micron particles, which is

appropriate for intravenous delivery.

Figure 2. Scanning electron microscope picture of HP-PLGA: �10k (left),

�45k (right).

440 Y. Zheng et al.

Assessing the encapsulation efficiency of hematoporphyrin

The corresponding encapsulation efficiency of HP in the

HP-PLGA-MBs was 63.50%� 1.26%. The HP loading effi-

ciency of the HP-PLGA-MBs was 2.15%� 0.13% (w/w).

In vitro release properties of HP-PLGA-MBs

Figure 3 compares the release profiles of HP-PLGA-MBs

with/without low-frequency ultrasound sonication. The

release profiles of HP-PLGA-MBs with/without sonication

Figure 5. Contrast enhanced tumor at 16, 18, 20, 22, 24 and 26 s after the injection of HP-PLGA-MBs, and the Time–intensity curve showed the contrast-

enhanced ultrasound imaging of H22 hepatocellular carcinoma.

Figure 6. H22 tumor growth curves for control mice treated by Saline only

and mice treated by HP-PLGA-MBs-US, PLGA-MBs-US, HP-US, Saline-

US. It showed that the tumor regression rate in the HP-PLGA-MBs-US

group was faster than that in the HP-US group (p5 0.05) and in the

control group (p5 0.01) (*p5 0.05; **p5 0.01).

Figure 4. The microbubbles in vitro in a 1 mm plastic tube observed by

ultrasound contrast imaging: (A) saline and (B) HP-PLGA-MBs.

Figure 3. Cumulative release of HP from HP-PLGA-MBs with sonication

(ultrasound) and without sonication (control) in PBS.

Hematoporphyrin encapsulated PLGA microbubble 441

were measured and compared to evaluate the effects of

sonication on their release properties. Each release profile

is represented by the percentage of released hemotopor-

phyrin as a function of the release time. The results from

Figure 3 show that the release rate of hematoporphyrin

with sonication is faster than that without sonication.

Approximately 90% of the hematoporphyrin was released

after 2 days, but for the HP-PLGA-MBs without sonication

less than 65% was released. We also determined the values

of the time for 50% of the hematoporphyrin to be released

(t1/2) for the HP-PLGA-MBs. The average t1/2 values for

HP-PLGA-MBs with/without sonication were 3 and 24 h,

respectively. This result implied that the HP-loaded bub-

bles can be controllably disrupted with US, and the HP can

be released and deposited at the target sites under US

image guidance. The mechanism for the ultrasound-trig-

gered drug release from PLGA microbubbles might be the

degradation of PLGA caused by the sonication (El-Sherif

et al., 2004). El-Sherif et al. found that the rate of degrada-

tion is dependent not only on exposure to ultrasound, but

also on the frequency at which the microcapsules were

insonated, and the method by which the capsules were

fabricated (El-Sherif et al., 2004). This information is critical

in understanding future work on the ultrasound triggered

drug delivery pattern. It is hypothesized that the fre-

quency that causes the fastest degradation rate of cap-

sules will also produce an increase in the release rate

of the adsorbed drug. Further studies are required to be

done in order to find the best ultrasound frequency and

preparation method for the encapsulation of the

sonosentilizers.

Figure 7. (a) The apoptosis index (AI) of H22 tumor cells in each group.

There is no significant difference for the apoptotic index (AI) between the

control group and the Saline-US group (p4 0.05). In contrast, the AI for

the sonicated tumor in the HP-PLGA-MBs-US group and HP-US group is

significantly bigger than in the saline group (p5 0.01). And the AI for the

tumor in the HP-PLGA-MBs-US group is bigger than that in the HP-US

group (p5 0.05). (*p5 0.05; **p5 0.01; �p5 0.05 comparing with HP-

US). (b) The sonosensitizer caused H22 tumor cells apoptosis in the tumor

bearing mice (Tunel method, �400). The nucleus of the apoptosis cells

were dyed brown. Much more apoptosis cells were observed in the HP-US

and HP-PLGA-MBs-US group: (A): control; (B): Saline-US; (C): PLGA-

MBs-US; (D): HP-US; (E): HP-PLGA-MBs-US.

Table 3. The proliferating index of H22 tumor cell in each group.

Control Saline-

US

PLGA-

MBs-US

HP-US HP-PLGA-

MBs-US

PI (%) 90.09� 3.95 86.94� 4.92 84.79� 6.28 54.33� 7.80* 37.83� 6.34**

Note: *p5 0.01; **p5 0.05.

Table 1. Tumor inhibition ratios of the tumor weight (%).

Control Saline-US PLGA-MBs-US HP-US HP-PLGA-MBs-US

Tumor weight (g) 1.48� 0.23 1.38� 0.20 1.36� 0.30 0.97� 0.19 0.68� 0.23

Tumor inhibition ratios (%) 0 6.51 7.89 34.36* 53.86**

Note: *p5 0.01; **p5 0.05.

Table 2. The apoptosis index of H22 tumor cells in each group.

Control Saline-US PLGA-MBs-US HP-US HP-PLGA-MBs-US

AI (%) 16.14� 3.93 19.22� 5.06 21.02� 5.87 36.71� 7.28* 52.82� 5.98**

Note: *p5 0.01; **p5 0.05.

442 Y. Zheng et al.

In vitro ultrasound contrast enhancement

The HP-PLGA-MBs bubbles could well enhance ultra-

sound imaging under default settings for clinical harmonic

imaging (Figure 4). The contrast-enhanced image intensity

after the injection of HP-PLGA-MBs was significantly

bigger than that of water, which was 112.20� 6.58 and

0.80� 0.45, respectively (p5 0.01). This result implied

that the HP-PLGA-MBs could be used for clinical ultra-

sound contrast imaging with proper development.

In vivo ultrasound contrast enhancement of mouse tumour

Tumour accumulation of HP-PLGA-MBs after systemic

injections of HP-PLGA-MBs was clearly manifested by

increased echogenicity of tumour ultrasound images

(Figure 5). This implies that the ultrasonic exposure

could be guided by the HP-PLGA-MBs enhanced ultra-

sound tumour imaging. As soon as the HP-PLGA-MBs

arrive at the tumour place, the ultrasonic exposure

should be applied. The HP was released locally, which

was considered be able to improve the efficiency of che-

motherapy and alleviate the side effects.

In vivo tumour therapy

The unsonicated tumour in the Saline group grew with the

same rate as the tumours in Saline-US group (p4 0.05).

In contrast, the sonicated tumour appeared resolved after

three treatments in the HP-PLGA-MBs-US group and HP-

US group. While the tumour regression rate in the HP-

PLGA-MBs-US group is faster than that in the HP-US

group (p5 0.05) and in the control group (p5 0.01)

(Figure 6). The results of the tumour inhibition ratios of

the tumour weight are presented in Table 1. These data

indicate that without HP, the simple ultrasound or simple

PLGA-MBs had no significant affect on the tumour growth.

However, the HP-PLGA-MBs plus US is more effective than

that of pure HP plus US.

There is no significant difference for the AI between the

control group and the Saline-US group (p4 0.05). In con-

trast, the AI for the sonicated tumour in the HP-PLGA-MBs-

US group and HP-US group is significantly bigger than in

the saline group (p5 0.01). And the AI for the tumour in the

HP-PLGA-MBs-US group is bigger than that in the HP-US

group (p5 0.05) (Table 2, Figure 7(a) and (b)).

There is no significant difference for the he proliferating

index (PI) between the control group and the Saline-US

group (p4 0.05). In contrast, the PI for the sonicated

tumour in the HP-PLGA-MBs-US group and HP-US group

is significantly lower than in the saline group (p5 0.05).

And the PI for the tumour in the HP-PLGA-MBs-US group

is lower than that in the HP-US group (p5 0.05) (Table 3,

Figure 8(a) and (b)).

Since the AI of the tumours is positively related to the

tumour regression rate and the proliferating index is neg-

atively related to the tumour regression rate, these data

indicated that the encapsulation of the sonosensitizer

inside the high molecular microbubbles, the HP-PLGA-

MBs, could significantly improve the therapeutic effects

of the sonosensitizer in tumour-bearing mice model.

Conclusion

In conclusion, we successfully constructed HP-PLGA-MBs

composed of ultrasound-responsive polymeric microbub-

bles for systemic contrast-enhanced ultrasound imaging

diagnosis, and sonosensitizers inside the microbubbles

for sonodynamic therapy. The application of HP-PLGA-

MBs in vivo can significantly inhibit the growth of mice

Figure 8. (a) The proliferating index (PI) of H22 tumor cells in each group.

There is no significant difference for the he Proliferating index (PI)

between the control group and the Saline-US group (p4 0.05). In con-

trast, the PI for the sonicated tumor in the HP-PLGA-MBs-US group and

HP-US group is significantly lower than in the saline group (p5 0.05).

And the PI for the tumor in the HP-PLGA-MBs-US group is lower than

that in the HP-US group (p5 0.05). (*p5 0.05; **p5 0.01; �p5 0.05

comparing with HP-US). (b) The sonosensitizer inhibited the proliferating

of the H22 tumor bearing mice (�400). The nucleus of the proliferating

cells were dyed brown. (PCNA stain, �400). Much less proliferating cells

were observed in the HP-US and HP-PLGA-MBs-US group: (A): control

(Saline); (B): Saline-US; (C): PLGA-MBs-US; (D): HP-US; (E): HP-PLGA-

MBs-US.

Hematoporphyrin encapsulated PLGA microbubble 443

tumours. Meanwhile, HP-PLGA-MBs can still maintain

adequate acoustic properties that are required to act as

an ultrasound contrast agent. Thus, the dual-functional

ultrasound microbubbles hold a great potential for ultra-

sound-guided sonodynamic tumour therapy. This simple

and highly efficient theranostic agent would provide a

novel strategy for the sonodynamic therapy of tumour.

Limitation

In this study, we focused on the demonstration of the fea-

sibility of a sonosensitizer loaded multi-functional contrast

agent, while we did not do the examination of the drug

distribution and the side effect, which is a limitation of

this study. This should be done in our future work.

Acknowledgements

This research is partialy supported by National Natural

Science Foundation of China (No. 81130025, 30900371,

81071158, 81071157), National Key Basic Research

Program (No. 2011CB707900), Foundation for Sci and

Tech Research Project of Chongqing (2010AB50952) and

Scientific Research Foundation for Returned Scholars

(No. 2010609).

Declaration of interest

The authors report no conflicts of interest. The authors alone

are responsible for the content and writing of the article.

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