The evaluation of cellular uptake efficiency and tumor-targeting ability of MPEG–PDLLA micelles: effect of particle size

Abstract

In this study, we successfully prepared MPEG–PDLLA polymer micelles with two different particle sizes, A and B. DLS and TEM assays demonstrated that the particle sizes of the polymer micelles A and polymer micelles B were about 25 nm and 150 nm respectively. The stability of the MPEG–PDLLA polymer micelles in vitro revealed that the free Cy5.5 dye had been successfully loaded into the polymer micelles as a fluorescence marker and the fluorescence wasn’t quenched until 72 h. The cellular uptake of the polymer micelles was time-dependent and micelles A (particle size 25 nm) showed a higher efficiency to be internalized into the cytoplasm of MCF-7 cells than micelles B (particle size 150 nm). Furthermore, in vivo and in vitro biodistribution and tumor-targeting of polymer micelles MPEG–PDLLA were investigated in female MCF-7 tumor-bearing balb/cA-nu mice with an IVIS imaging system. The results showed that polymer micelles A (particle size 25 nm) had a time dependent biodistribution and tumor site accumulation in mice bearing MCF-7 tumors. Meanwhile, the NIR fluorescence intensity of polymer micelles B (particle size 150 nm) in tumor sites showed a pattern of a rise, a peak and then a decline. What’s more, the distribution of the polymer micelles in the tissue slices demonstrated the same results. Consequently, the results indicated that the micelles with a smaller particle size (25 nm) could be more efficiently internalized into cells and increase the enhanced permeation and retention (EPR) effect in tumor tissue. Therefore, a reasonable small size of micelles may be a key factor for a high-performance anti-cancer drug delivery system.

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Introduction

Nowadays, more and more people are suffering from cancer, but the traditional anti-cancer drugs have a lot of drawbacks, such as being poorly water-soluble,^1,2 having side effects and multidrug resistance.^3,4 Nanotechnology highlights the hope for cancer therapy as a large number of nanocarriers are used in the clinical setting.^5–8

In recent years, amphiphilic block copolymers have attracted significant attention as a means of delivering anti-cancer drugs because amphiphilic block copolymers consist of hydrophilic and hydrophobic segments, which have the ability to form nanocarriers and self-assemble in aqueous solutions.^9–11 Nanocarriers can not only transport anticancer agents, but also have some advantages such as long-circulation time, high cellular uptake efficiency and can preferentially reach tumor sites.^12,13 What’s more, they have shown different cellular uptake efficiency and tumor targeting abilities depending on their sizes.^14–16 It has been proven that particle sizes ranging from 25 to 50 nm are suitable for achieving high cellular uptake efficiency as the cellular uptake of nanocarriers is regulated by membrane tension, the optimal radius for endocytosis is on the order of 25–50 nm.^17–20 Additionally, nanocarriers in the size range of 10–200 nm are highly recommended for tumor accumulation, owing to the advantages of reducing clearance from the reticuloendothelial system (RES), and increasing tumor accumulation through the enhanced permeation and retention (EPR) effect.^8,21

Among amphiphilic block copolymers,^22–24 the monomethoxy poly(ethylene glycol)–poly(D,L-lactic acid) copolymer (MPEG–PDLLA) has sparked interest as a drug delivery carrier.²⁵ MPEG–PDLLA consists of polyethylene glycol (MPEG) and poly(D,L-lactide), which are FDA-approved non-cytotoxic, non-immunogenic polymers and have been widely used as a long-circulating agent to improve biocompatibility, stability and have a good record of offering great potential for controlled release.^26,27 Samyang’s proprietary polymeric micelle technology utilizing PTX loaded MPEG–PDLLA (M_n = 3765) micelles (Genexol-PM®) for cancer chemotherapy was applied in the clinic and approved in Korea in 2006.²⁸ In addition, the study of MPEG–PDLLA is carrying on.^29–32 However, the study on the particle size of the nanocarrier MPEG–PDLLA still needs to develop as the particle size determines the mechanism and rate of cellular uptake of nanocarriers and their ability to permeate through tissue.³³ A particle size less than 5 nm is rapidly cleared from the circulation through renal clearance or extravasation, and as particle size increases to 15 μm, the accumulation occurs primarily in the liver, spleen and bone marrow which may cause certain toxicities.^34,35 It is necessary to study the influence of particle size of the nanocarrier MPEG–PDLLA immediately and determine which size of MPEG–PDLLA is promising as a nanocarrier.

In this study, we aimed to evaluate the cellular uptake efficiency and tumor-targeting ability of MPEG–PDLLA micelles with two different particle sizes, 25 nm and 150 nm. As MPEG–PDLLA has no fluorescent group, we chose a Cy5.5–NHS ester as a fluorescent marker.^36–39 The Cy5.5–NHS ester is a fluorescent marker in the near infrared region (NIR) spectrum, which is suitable for small animal in vivo live imaging instead of radioactive element imaging.^40–42 We used two methods to get MPEG–PDLLA micelles with two different particle sizes. The cellular uptake efficiency of the micelles was investigated in MCF-7 cells. Then we detected the biodistribution and tumor-targeting of the MPEG–PDLLA micelles with different particle sizes by an IVIS imaging system. This study is the first time an IVIS imaging system has been used to detect the biodistribution and tumor-targeting of MPEG–PDLLA with two different particle sizes, which was more sensitive, cost-effective, easy-to-use, and avoided the use of radiopharmaceuticals. We could easily get the real-time difference of the biodistribution between MPEG–PDLLA with different particle sizes through the IVIS imaging system, and the results could be intuitive to see whether the same nanocarrier with different particle sizes has a different delivery efficiency, and which particle size of the MPEG–PDLLA micelles is suitable for delivering anti-cancer drugs.

Materials and experimental methods

Materials

Monomethoxy-poly(ethylene glycol) with a molecular weight of 2000 (MPEG 2000), stannous octoate (Sn(Oct)₂), dicyclohexylcarbodiimide (DCC) and N-(tert-butoxycarbonyl)-L-alanine (BOC-L-AlA) were obtained from the Sigma Aldrich company. D,L-Lactide was purchased from Beijing Jiankai Corporation in China. Anhydrous ethanol was purchased from the Shanghai Aladdin company. The Cy5.5–NHS ester was purchased from the Beijing Fanbobiochemicals company. All the materials used in this article were of analytical grade and used as received.

An MCF-7 cell line was obtained from the American Type Culture Collection (ATCC; Rockville, MD) and grown in RPMI DMEM media with 10% FBS and 1% antibiotics. The cell culture was maintained in a 37 °C incubator with a humidified 5% CO₂ atmosphere.

Balb/cA-nu mice used for in vivo and in vitro fluorescence imaging were purchased from the HFK Bio-Technology. Co., LTD (Beijing, China). Throughout the experiment, the animals were housed at a temperature of 20 ± 2 °C, relative humidity of 50–60%, and with 12 h light–dark cycles. All animal care and experimental procedures were conducted according to Institutional Animal Care and Use guidelines.

Synthesis of MPEG–PDLLA–NH₂

The MPEG–PDLLA copolymer (2000–1765) was synthesized by ring opening polymerization. 17.65 g D,L-lactide and 20 g MPEG 2000 were put into a dried glass reactor already flame-dried and purged three times by nitrogen. Then 0.3% stannous octoate (0.113 g) was added in an environment of nitrogen. The polymerization started in an oil bath at 150 °C for 9 h. After the completion of the reaction, the products were dissolved in 50 mL ethanol, then precipitated in 500 mL cold n-pentane and filtered three times. The final copolymer was kept in a vacuum at 35 °C for 48 h.⁴³

The diblock copolymer MPEG–PDLLA was converted into BOC-L-AlA as follows: a nitrogen-purged flask containing 10.0 g MPEG–PDLLA and 4.0 g BOC-L-AlA dissolved in 100 mL anhydrous CH₂Cl₂ was treated with a solution of 3.0 g DCC for 72 h at 25 °C. Dicyclohexylurea was removed by vacuum filtration. The filtrate was washed with 100 mL saturated aqueous NaHCO₃ and 100 mL distilled water three times. Then the copolymer was obtained by removing the organic solvents in a vacuum by a rotary evaporator.⁴⁴

MPEG–PDLLA–NH₂ was synthesized by removing the tert-butoxycarbonyl end group from MPEG–PDLLA–BOC⁴⁵ as follows: 5 g MPEG–PDLLA–BOC was dissolved in 50 mL CH₂Cl₂. The solution was cooled to 0 °C and treated with 15 mL trifluoroaceticacid (TFA) for 2.5 h in an atmosphere of nitrogen. TFA was then removed in a vacuum, the residue was dissolved in 40 mL chloroform and washed with 100 mL saturated aqueous NaHCO₃ and 100 mL distilled water three times. Finally the copolymer was obtained by removing the organic solvents in a vacuum by a rotary evaporator.

Characterization of MPEG–PDLLA–NH₂

The diblock copolymer was characterized by ¹H NMR spectra (Varian 400 spectrometer, Varian, USA), FTIR (NICOLET 200SXV, Nicolet, USA) and GPC (Agilent 110 HPLC, USA).⁴⁶

Synthesis of the fluorescence marker MPEG–PDLLA–Cy5.5

MPEG–PDLLA–Cy5.5 was synthesized by reacting the Cy5.5–NHS ester (0.5 mg) with MPEG–PDLLA–NH₂ (100 mg) dissolved in 5 mL dimethylsulfoxide at room temperature for 24 h.^47,48 Then using a dialysis bag with the molecular mass cutoff of 2 kDa to remove free Cy5.5–NHS and dimethylsulfoxide for 48 h. The final product was free-dried and stored at −20 °C in the dark until used.

Preparation of the MPEG–PDLLA micelles

In this research, we used two methods to prepare MPEG–PDLLA micelles. One was a thin-film hydration method.^29,49 Briefly, 30 mg MPEG–PDLLA and 1 mg MPEG–PDLLA–Cy5.5 were dissolved together in 4 mL anhydrous ethanol to prepare a polymer solution. Then a rotary evaporator was used to remove anhydrous ethanol in a vacuum at 37 °C for 2 h. Finally, 4 mL of distilled water was added to prepare micelles at 60 °C and filtered with a syringe filter (pore size: 220 nm) (Millex-LG, Millipore Co., USA). All operations were conducted in the dark.

The other method was an ethanol injection method.^50,51 30 mg MPEG–PDLLA and 1 mg MPEG–PDLLA–Cy5.5 were dissolved together in 4 mL of ethanol. The mixed polymer in anhydrous ethanol was slowly added to 4 mL of distilled water and stirred at 60 °C to remove anhydrous ethanol for about 5 h. Then micelles were filtered with a syringe filter (pore size: 220 nm) (Millex-LG, Millipore Co., USA). All operations were conducted in the dark.

The concentration of the free dye group (control group) was 1.25 μg mL⁻¹ which was same as the concentration of the polymer micelles. The free Cy5.5 dye was hydrophobic, so we used 1 mL DMSO to dissolve 1 mg Cy5.5 dye to get a 1 mg mL⁻¹ stock solution. Then we took a 1.25 μL stock solution (1 mg mL⁻¹) and diluted it with saline to 1 mL to get the free dye.

Characterization of the MPEG–PDLLA micelles

The particle size of the MPEG–PDLLA polymer micelles was measured by dynamic light scattering (DLS) (Nano-ZS90, Malvern, UK). The morphology of the polymer micelles was observed using transmission electron microscopy (TEM) (H-6009IV, Hitachi, Japan). Before using TEM to observe, samples were placed on a carbon-coated copper grid, and negatively stained by phosphotungstic acids.⁵²

The stability of the MPEG–PDLLA micelles with a Cy5.5 fluorescence marker

The stability of the MPEG–PDLLA polymer micelles was measured by a fluorescence spectrophotometer (Perkin Elmer, USA).⁵³ We used the fluorescence spectrophotometer to observe the fluorescence intensity at 5 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h and 72 h after the polymer micelles were prepared.

Cellular imaging

We used MCF-7 breast cancer cells to investigate the uptake of the MPEG–PDLLA polymer micelles.^54,55 First, MCF-7 breast cancer cells were seeded in 6-well plates (2 × 10⁵) and incubated for 24 h. Then RPMI DMEM media were used with 2 mL of 1.25 μg mL⁻¹ polymer micelles and 1% antibiotics (RPMI DMEM media with 2 mL of 1% antibiotics was used as the control group) to replace the RPMI DMEM media with 10% FBS and 1% antibiotics per well. Next they were incubated for an additional 30 min (1 h, 2 h, 4 h, and 6 h). After removing the supernatant, the cells were fixed with 70% ETOH for 15 min and then DAPI was added for a 10 min incubation. Subsequently, the cells were washed 3 times with PBS and sealed with glycerine. The cellular uptake of the polymer micelles was determined using confocal laser scanning microscopy (CLSM) from Germany Leica Corporation. The quantitative data was analyzed by flow cytometry from USA BD Bioscience Corporation.

In vivo and in vitro fluorescence imaging

An in vivo and in vitro fluorescence imaging study was performed in female MCF-7 tumor-bearing balb/cA-nu mice.^56,57 Tumors were initially established by injecting a mixture of 1 × 10⁶ MCF-7 cells subcutaneously. When the tumors reached a volume of 200 mm³, the biodistribution of the MPEG–PDLLA polymer micelles was studied by injecting 100 μL of 1.25 μg mL⁻¹ micelles (free Cy5.5) intravenously through the tail vein of mice bearing MCF-7 tumors. These were imaged at 5 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h and 72 h after injection with an IVIS imaging system (Perkin Elmer, USA). Dye accumulation and retention in tumors were evaluated quantitatively by calculating the ROI values.

The tumor-bearing mice were killed at 2 h, 8 h, 24 h, 48 h and 72 h after the polymer micelles were injected, and then the livers, hearts, spleens, lungs, kidneys and tumors were collected for isolated organ imaging to estimate the tissue distribution of the micelles. We evaluated the fluorescence intensity of tissues quantitatively by calculating the ROI values.

Tissue slices

The liver, heart, spleen, lung, kidney and tumor tissue were used to make tissue slices which were harvested at 2 h, 8 h, 24 h, 48 h and 72 h after the MPEG–PDLLA micelles were injected.⁵⁸ In detail, an embedding medium was used to fix the tissues and get tissue slices using a frozen section machine. Then DAPI was added for 5 min and they were sealed with glycerine. A confocal laser scanning microscope from Germany Leica Corporation was used to observe the fluorescence intensity of the tissues.

Statistics

Statistical analysis was performed using a Student’s t-test or one-way analysis of variance (ANOVA). All data were expressed as the mean value ± SD. P-values less than 0.05 were considered to be statistically significant.

Results and discussion

Characterization of MPEG–PDLLA–NH₂

In this study, we successfully synthesized the MPEG–PDLLA–NH₂ block copolymer and reacted it with the fluorescence marker Cy5.5 dye. The synthesis route is illustrated in Fig. 1. In detail, the MPEG–PDLLA copolymer was synthesized by ring opening polymerization, then the BOC-L-AlA was converted into MPEG–PDLLA. Our target product was obtained by removing the tert-butoxycarbonyl end group from MPEG–PDLLA–BOC. MPEG–PDLLA, MPEG–PDLLA–BOC and MPEG–PDLLA–NH₂ were characterized by a ¹H NMR spectrum, and the molecular weights and polydispersity of the copolymer were determined by GPC. The molecular weight data of MPEG–PDLLA, MPEG–PDLLA–BOC and MPEG–PDLLA–NH₂ are summarized in Table 1.

Fig. 1 The synthesis route of MPEG–PDLLA–Cy5.5.

Table 1 The molecular weight data of MPEG–PDLLA, MPEG–PDLLA–BOC and MPEG–PDLLA–NH₂

Sample	Mn^a	Mn^b	Mn^c	Mw^c	Mw^c/Mn^c
a Theoretical molecular weight.b Calculated from ^1H NMR data.c Measured by GPC.
MPEG–PDLLA	3765	3821	3360	4152	1.24
MPEG–PDLLA–BOC	3850	4315	4147	4649	1.12
MPEG–PDLLA–NH2	3950	3980	4234	4902	1.16

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From the results of the ¹H NMR spectra in Fig. 2, we can see four peaks marked with letters from a to d in Fig. 2(A) MPEG–PDLLA, a at 3.62 ppm (CH₃O–), b at 3.63 ppm (–CH₂CH₂O–), c at 5.15 ppm (–COCH(CH₃)O–), and d at 1.55 ppm (–CH₃). In the ¹H NMR spectrum in Fig. 2(B), the peak marked with letter e (1.39 ppm) represents the tert-butoxycarbonyl group, demonstrating that the BOC-terminated MPEG–PDLLA block polymer was successfully synthesized. The peak e (1.39 ppm) disappeared completely in Fig. 2(C), since we eliminated the tert-butoxycarbonyl group and obtained MPEG–PDLLA–NH₂.

Fig. 2 The ¹H NMR spectra of (A) MPEG–PDLLA, (B) MPEG–PDLLA–BOC, and (C) MPEG–PDLLA–NH₂ in CDCl₃.

Analysis by GPC revealed the retention times of MPEG–PDLLA, MPEG–PDLLA–BOC and MPEG–PDLLA–NH₂ in Fig. 3. Fig. 3(A) MPEG–PDLLA demonstrates a single peak with polydispersity of 1.24 and M_n of 3360. Fig. 3(B) shows a shift to a later retention time for MPEG–PDLLA–BOC, which is consistent with an increase in M_n of 4147. Fig. 3(C) MPEG–PDLLA–NH₂ reveals an almost similar shape and the same position for the retention time as Fig. 3(B), indicating that the polymeric structure was kept unchanged and the molecular weight and distribution changed very little.

Fig. 3 The retention time of (A) MPEG–PDLLA, (B) MPEG–PDLLA–BOC, and (C) MPEG–PDLLA–NH₂ measured by GPC.

The IR spectra of MPEG–PDLLA, MPEG–PDLLA–BOC and MPEG–PDLLA–NH₂ are shown in Fig. 4. From Fig. 4(A), the absorption peak at 1188.76 cm⁻¹ and 1455.00 cm⁻¹ can be seen to belong to the MPEG block. The peak at 1755.10 cm⁻¹ was characteristic of the PDLLA block. The absorption peaks at 1526.61 cm⁻¹ and 3324.82 cm⁻¹ were attributed to the νNH stretch vibration in Fig. 4(B), indicating that the tert-butoxycarbonyl group was added into the MPEG–PDLLA block polymer successfully. A νNH stretch vibration was also found at 3327.90 cm⁻¹ and 1535.68 cm⁻¹ in Fig. 4(C).

Fig. 4 The IR spectra of (A) MPEG–PDLLA, (B) MPEG–PDLLA–BOC, and (C) MPEG–PDLLA–NH₂.

Characterization of the MPEG–PDLLA micelles

In this study, we used two methods to prepare the MPEG–PDLLA polymer micelles with different particle sizes. The schematic illustration of the preparation of the MPEG–PDLLA polymer micelles and a flow diagram of the experiment is shown in Fig. 5.

Fig. 5 The schematic illustration of the preparation of the MPEG–PDLLA polymer micelles and a flow diagram of the experiment.

We used a thin-film hydration method to prepare MPEG–PDLLA micelles. During the procedure, the MPEG–PDLLA copolymer was distributed as a homogenous, amorphous thin-film. Then the copolymer self-assembled to a core–shell structure in the water system. The polymer micelles were obtained after filtering with a syringe filter (pore size: 220 nm). As shown in Fig. 6(A), the particle size of the MPEG–PDLLA polymer micelles measured by dynamic light scattering (DLS) was 24.59 ± 1.16 nm. In the morphology study, the same diameter for the MPEG–PDLLA polymer micelles was observed in the TEM image.

Fig. 6 The particle size distribution and TEM (inset image) of the MPEG–PDLLA polymer micelles, (A) MPEG–PDLLA polymer micelles (particle size 25 nm), and (B) MPEG–PDLLA polymer micelles (particle size 150 nm).

According to Fig. 6(B), the particle size of MPEG–PDLLA polymer micelles prepared by ethanol injection method was 150.27 ± 1.62 nm. The TEM image showed the same diameter for the MPEG–PDLLA micelles.

The stability of the MPEG–PDLLA micelles with a Cy5.5 fluorescence marker

To investigate the stability of the MPEG–PDLLA polymer micelles in vitro by this system, we used a fluorescence spectrophotometer to observe the fluorescence intensity of the polymer micelles at 5 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h and 72 h after the polymer micelles were prepared as shown in Fig. 7. From the results, the fluorescence intensity of the free Cy5.5 dye, polymer micelles A (particle size 25 nm) and polymer micelles B (particle size 150 nm) followed the same trend and didn’t decrease. It demonstrates that the fluorescence wasn’t quenched until 72 h. The fluorescence intensity of the free Cy5.5 dye was lower than that of the polymer micelles A (particle size 25 nm) and the polymer micelles B (particle size 150 nm), indicating that the free Cy5.5 dye had been successfully loaded into the polymer micelles.

Fig. 7 The fluorescence intensity of the polymer micelles A (particle size 25 nm), the polymer micelles B (particle size 150 nm) and free Cy5.5 dye in vitro at 5 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h and 72 h. The data points represent mean values and the bars represent standard deviation (n = 3).

Cellular imaging

We investigated the cellular uptake efficiency of the MPEG–PDLLA polymer micelles over time qualitatively by using confocal laser scanning microscopy (CLSM). Here we compared the cellular uptake efficiency between the polymer micelles A (particle size 25 nm) and the polymer micelles B (particle size 150 nm) at 30 min, 1 h, 2 h, 4 h, and 6 h after adding the polymer micelles. The result showed that micelles A (particle size 25 nm) could more efficiently penetrate into MCF-7 cells and that the cellular uptake efficiency was time-dependent. This result is consistent with previous studies^17–20 as the cellular uptake of nanocarriers are regulated by membrane tension, the optimal radius for endocytosis is on the order of 25–50 nm.

Fig. 8(A) shows the pictures of MCF-7 cells incubated with polymer micelles A (particle size 25 nm). After a 0.5 h incubation with polymer micelles A (particle size 25 nm), the column of Cy5.5 was almost black. It could be seen that DAPI stained nuclei were circumvented by the MPEG–PDLLA polymer micelles A at 1 h, indicating that the polymer micelles A (particle size 25 nm) had been internalized into the cytoplasm of MCF-7 cells. Also the fluorescence intensity increased with time.

Fig. 8 The cellular uptake of the (A) polymer micelles A (particle size 25 nm), and (B) polymer micelles B (particle size 150 nm) at 30 min, 1 h, 2 h, 4 h and 6 h. (Column 1: Cy5.5 channels showing red fluorescence from the MPEG–PDLLA polymer micelles distributed in the cytoplasm. Column 2: DAPI channels showing blue fluorescence from nuclei. Column 3: Merged channels of Cy5.5 and DAPI.) (C) The quantitative data analyzed using flow cytometry. The data points represent mean values and the bars represent standard deviation (n = 3). The double star (**) indicates P < 0.01.

From Fig. 8(B), we can also see that the polymer micelles B (particle size 150 nm) were internalized into the cytoplasm of MCF-7 cells and that the fluorescence intensity increased with the time. It was noted that the red fluorescence intensity in Fig. 8(A) is higher than that in Fig. 8(B), demonstrating that cellular uptake was easier for the polymer micelles with a smaller particle size.

Cellular uptake of the MPEG–PDLLA polymer micelles with different particle sizes at different times was studied quantitatively by measuring the percentage of internalized Cy5.5 in MCF-7 cells in Fig. 8(C). After a 0.5 h incubation with the polymer micelles, 20.26 ± 2.81% of the polymer micelles A (particle size 25 nm) and 18.37 ± 1.02% of the polymer micelles B (particle size 150 nm) were internalized into MCF-7 cells. After a 6 h incubation, 56.02 ± 2.48% of the polymer micelles A (particle size 25 nm) and 48.43 ± 2.08% of the polymer micelles B (particle size 150 nm) were internalized into the MCF-7 cells respectively. The results indicated that between 30 min and 6 h, the polymer micelles showed time-dependent cellular uptake and the cellular uptake efficiency of the polymer micelles A (particle size 25 nm) was higher than that of the polymer micelles B (particle size 150 nm), demonstrating the same results as Fig. 8(A) and (B).

In vivo fluorescence imaging

In vivo real-time biodistribution and tumor-targeting of the MPEG–PDLLA polymer micelles in MCF-7 tumor-bearing mice were evaluated through NIR florescence imaging with an IVIS imaging system at 5 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h and 72 h after being injected intravenously with the polymer micelles. The diagnosis profiles of the polymer micelles A (particle size 25 nm), polymer micelles B (particle size 150 nm) and free Cy5.5 dye were clearly visualized by monitoring real-time NIR fluorescence intensity in the whole body as shown in Fig. 9.

Fig. 9 In vivo time-dependent fluorescence images of MCF-7 tumor-bearing mice at 5 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, 24 h, 48 h and 72 h after being injected intravenously with the polymer micelles. The color bar from red to blue indicates the change in fluorescence signal intensity from low to high. (A) The polymer micelles A (particle size 25 nm), (B) polymer micelles B (particle size 150 nm), (C) free Cy5.5 dye, and (D) the quantitative fluorescence intensity measured using the ROI value. The data points represent mean values and the bars represent standard deviation (n = 3). The double star (**) indicates P < 0.01.

According to Fig. 9(A), the polymer micelles A (particle size 25 nm) had a time dependent biodistribution and tumor accumulation in mice bearing MCF-7 tumors. Between 5 min and 72 h post-injection, the NIR fluorescence signals of the polymer micelles A (particle size 25 nm) increased within tumors, although decreasing in the whole body, which may be due to specific targeting of tumor cells. We quantified the fluorescence intensity in the tumor using the ROI value in Fig. 9(D). The ROI value showed that the fluorescence intensity at the tumor site didn’t decrease until 72 h.

From the results in Fig. 9(B), the administration of the polymer micelles B (particle size 150 nm) resulted in a fluorescence signal which gradually decreased with time. Between 5 min and 24 h after injection, the difference in the fluorescence intensity at the tumor site was smaller for MCF-7 tumor-bearing mice injected with either the polymer micelles A (particle size 25 nm) or the polymer micelles B (particle size 150 nm). However, at 48 h post-injection, the polymer micelles A (particle size 25 nm) showed a stronger fluorescence intensity at the tumor site than the polymer micelles B (particle size 150 nm). The same results are shown in Fig. 9(D), demonstrating that the enhanced permeation and retention (EPR) effect was affected by the particle size.

In this study, we used the group of free Cy5.5 dye as a control group. From Fig. 9(C), the fluorescence signal at the tumor was weaker than the whole body all the time. The quantitative fluorescence intensity measured by the ROI value in Fig. 9(D) shows the same results, indicating that tumor accumulation of fluorescence was due to the polymer micelles.

In vitro fluorescence imaging

In this part, the MCF-7 tumor-bearing mice were killed at 2 h, 8 h, 24 h, 48 h and 72 h after the MPEG–PDLLA polymer micelles were injected intravenously, and then the livers, hearts, spleens, lungs, kidneys and tumors were isolated to estimate the tissue distribution of the polymer micelles using NIR florescence imaging with an IVIS imaging system.

The fluorescent signals of the MPEG–PDLLA polymer micelles in the deep organs were often underestimated by in vivo fluorescence imaging because of optical impedance by soft tissues. So the in vitro fluorescence imaging experiment shown in Fig. 10 was necessary for us to study the tissue distribution of the polymer micelles.

Fig. 10 In vitro imaging of the livers, hearts, spleens, lungs, kidneys and tumors excised from MCF-7 tumor-bearing mice at 2 h, 8 h, 24 h, 48 h and 72 h after being injected intravenously with the polymer micelles. The color bar from red to blue indicates the change in fluorescence signal intensity from low to high. (A) The polymer micelles A (particle size 25 nm), (B) polymer micelles B (particle size 150 nm), and (C) the free Cy5.5 dye. The quantitative fluorescence intensity of tissues measured using the ROI value. (D) The polymer micelles A (particle size 25 nm), (E) polymer micelles B (particle size 150 nm), and (F) the free Cy5.5 dye.

From the results of the tissue distribution of the polymer micelles A (particle size 25 nm) in Fig. 10(A), a significant enhancement of fluorescence signals was exhibited in tumors as the time extended, whereas the fluorescence intensity of the hearts, lungs and spleens decreased as the time extended. It was noted that, as the fluorescence intensity of the hearts showed only background to moderate signals, the acute cardiotoxicity of the heart may have decreased. The fluorescence intensity of the livers and kidneys from mice treated with polymer micelles A (particle size 25 nm) also displayed strong signals and peaked at 8 h post-injection, indicating that polymer micelles A (particle size 25 nm) were undergoing liver metabolism and renal excretion. In short, between 8 h and 72 h post-injection, the fluorescence intensity of the other tissues gradually decreased with time, but the fluorescence intensity at the tumor site remained at a relatively high level indicating that the polymer micelles A had a long circulation time and showed the enhanced permeability and retention (EPR) effect in the tumor. The quantitative fluorescence intensity of the tissues measured by the ROI value in Fig. 10(D) shows the same results.

According to Fig. 10(B), the tissues showed almost the same results as the polymer micelles A (particle size 25 nm) except for the tumors. The fluorescence intensity of the tumors showed a rise at first, followed by a decline. The tumor tissues had the strongest fluorescence intensity at 8 h post-injection and then decreased rapidly, demonstrating that the enhanced permeation and retention (EPR) effect was affected by particle size. The quantitative results measured using the ROI value are shown in Fig. 10(E) and are consistent with Fig. 10(B).

In this study, we used the group of free Cy5.5 dye as a control group. From Fig. 10(C) and (F), the fluorescence signal of all the tissues was weaker than the polymer micelle groups, indicating that the fluorescence of the tissue distribution was due to the polymer micelles.

Tissue slices

The distribution of MPEG–PDLLA polymer micelles was ever-changing with time after injection. In this part, we observed the fluorescence intensity using a confocal laser scanning microscope from the German Leica Corporation. The results of the images are shown in Fig. 11.

Fig. 11 Fluorescence images of the liver, heart, spleen, lung, kidney and tumor tissue slices from MCF-7 tumor bearing mice at 2 h, 8 h, 24 h, 48 h and 72 h after being injected intravenously with the polymer micelles. (A) The polymer micelles A (particle size 25 nm), (B) polymer micelles B (particle size 150 nm), and (C) the free Cy5.5 dye.

From the results of the tissue slice distribution of the polymer micelles A (particle size 25 nm) in Fig. 11(A), the fluorescence intensity of the tumor tissue slices rose with the time and maintained a fluorescence signal until 72 h post-injection. The fluorescence distribution in the livers and kidneys increased generally, and then decreased with time. The fluorescence signal of other tissue slices also decreased as the results showed for in vitro tissue fluorescence imaging.

Fig. 11(B) shows the tissue slice distribution of the polymer micelles B (particle size 150 nm). A seen from imaging, we found that the fluorescence intensity of the liver and kidney was a bit stronger than that for micelles A (particle size 25 nm), but the tumor tissue had the strongest fluorescence intensity at 8 h post-injection and then decreased rapidly, indicating that the particle size affected the enhanced permeation and retention (EPR) effect.

The results of the free Cy5.5 dye tissue slices are shown in Fig. 11(C). The fluorescence intensity of the tissue slices was weaker than that for the group of micelles A (particle size 25 nm) and polymer micelles B (particle size 150 nm). These tissue slice results are same as the results of in vivo and in vitro fluorescence imaging.

Conclusion

In this study, we successfully synthesized the diblock polymer MPEG–PDLLA–NH₂ and characterized it by ¹H NMR spectra, FTIR and GPC. We used hydrophobic Cy5.5 free dye reacting with the MPEG–PDLLA–NH₂ to get the fluorescence marker MPEG–PDLLA–Cy5.5. Then we separately used a thin-film hydration method and ethanol injection method to get two different particle sizes of polymer micelles. One was MPEG–PDLLA polymer micelles A (particle size 25 nm) and the other was MPEG–PDLLA polymer micelles B (particle size 150 nm). The polymer micelles were characterized by DLS and TEM and a fluorescence spectrophotometer. In addition, the cellular uptake experiment on MCF-7 cells in vivo suggested the cellular uptake was affected by particle size and time. Between 30 min and 6 h, the fluorescence intensity increased with the time, and the fluorescence intensity of the small particle size was stronger than that for the large one. Furthermore, we detected biodistribution and tumor-targeting of MPEG–PDLLA polymer micelles in MCF-7 tumor-bearing mice by in vivo and in vitro fluorescence imaging and tissue slices. The results showed that the fluorescence intensity of the free Cy5.5 dye was weaker than that of the group of the MPEG–PDLLA–Cy5.5 polymer micelles. It was noted that the fluorescence signal of the tumor tissue which was injected with polymer micelles A (particle size 25 nm) was stronger than that of the group of polymer micelles B (particle size 150 nm), demonstrating that the polymer micelles A (particle size 25 nm) accumulated more easily at the tumor site than the polymer micelles B (particle size 150 nm). Therefore, MPEG–PDLLA polymer micelles based on two different particle sizes demonstrated that the small particle sizes (25 nm) could have high cellular uptake efficiency and enhance the EPR effect in tumor tissue. In short, drug delivery systems with small particle sizes may have potential applications to deliver antitumor drugs. In the future, our research will focus on the relationship between the particle size and cellular uptake efficiency and tumor-targeting ability.

Conflict of interest

The authors have declared that no competing interest exists.

Acknowledgements

This work was financially supported by The National Science Fund for Distinguished Young Scholars (NSFC31525009), and Distinguished Young Scholars of Sichuan University (2011SCU04B18).

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Footnote

† Huang YX is co-first author with Hao Y.

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