Polymeric micelles and nanoemulsions as drug carriers: Therapeutic efficacy, toxicity, and drug resistance

    Polymeric Micelles and nanoemulsions as drug carriers: Therapeutic efficacy, toxicity, and drug resistance Roohi Gupta, Jill Shea, Courtney Scafe, Anna Shurlygina, Natalya Rapoport PII: DOI: Reference:

S0168-3659(15)00626-4 doi: 10.1016/j.jconrel.2015.06.019 COREL 7724

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

22 February 2015 10 June 2015 14 June 2015

Please cite this article as: Roohi Gupta, Jill Shea, Courtney Scafe, Anna Shurlygina, Natalya Rapoport, Polymeric Micelles and nanoemulsions as drug carriers: Therapeutic efficacy, toxicity, and drug resistance, Journal of Controlled Release (2015), doi: 10.1016/j.jconrel.2015.06.019

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

PT

POLYMERIC MICELLES AND NANOEMULSIONS AS DRUG CARRIERS: THERAPEUTIC EFFICACY, TOXICITY, AND DRUG RESISTANCE

SC

RI

ROOHI GUPTA1*, JILL SHEA2, COURTNEY SCAFE2, ANNA SHURLYGINA3, NATALYA RAPOPORT1**

*

AC CE P

TE

D

**Correspondence author Correspondence address: Natalya Rapoport, Ph.D., D.Sc. 36 S. Wasatch Dr., Room 3100 Department of Bioengineering University of Utah Salt lake City, UT 84112 [email protected]

MA

NU

1 – Department of Bioengineering, University of Utah 2 – Department of Surgery, University of Utah 3 – Institute of Physiology and Fundamental Medicine, Russian Academy of Medical Sciences, Siberian Branch.

Current address: Roohi Gupta, Ph.D. Fox Chase Cancer Center P0103, Department of Radiation Oncology 333 Cottman Avenue Philadelphia, PA 19111

ACCEPTED MANUSCRIPT Abstract

AC CE P

TE

D

MA

NU

SC

RI

PT

The manuscript reports the side-by-side comparison of therapeutic properties of polymeric micelles and nanoemulsions generated from micelles. The effect of the structure of a hydrophobic block of block copolymer on the therapeutic efficacy, tumor recurrence, and development of drug resistance was studied in pancreatic tumor bearing mice. Mice were treated with paclitaxel (PTX) loaded poly(ethylene oxide)-co-polylactide micelles or corresponding perfluorocarbon nanoemulsions. Two structures of the polylactide block differing in a physical state of micelle cores or corresponding nanodroplet shells were compared. Poly(ethylene oxide)co-poly(D,L-lactide) (PEG-PDLA) formed micelles with elastic amorphous cores while poly(ethylene oxide)-co-poly(L-lactide) (PEG-PLLA) formed micelles with solid crystalline cores. Micelles and nanoemulsions stabilized with PEG-PDLA copolymer manifested higher therapeutic efficacy than those formed with PEG-PLLA copolymer studied earlier. Better performance of PEG-PDLA micelles and nanodroplets was attributed to the elastic physical state of micelle cores (or droplet shells) allowing adequate rate of drug release via drug diffusion and/or copolymer biodegradation. The biodegradation of PEG-PDLA stabilized nanoemulsions was monitored by the ultrasonography of nanodroplets injected directly into the tumor; the PEGPDLA stabilized nanodroplets disappeared from the injection site within 48 hours. In contrast, nanodroplets stabilized with PEG-PLLA copolymer were preserved at the injection site for weeks and months indicating extremely slow biodegradation of solid PLLA blocks. Multiple injections of PTX-loaded PEG-PDLA micelles or nanoemulsions to pancreatic tumor bearing mice resulted in complete tumor resolution. Two of ten tumors treated with either PEG-PDLA micellar or nanoemulsion formulation recurred after the completion of treatment but proved sensitive to the second treatment cycle indicating that drug resistance has not been developed. This is in contrast to the treatment with PEG-PLLA micelles or nanoemulsions where all resolved tumors quickly recurred after the completion of treatment and proved resistant to the repeated treatment. The prevention of drug resistance in tumors treated with PEG-PDLA stabilized formulations was attributed to the presence and preventive effect of copolymer unimers that were in equilibrium with PEG-PDLA micelles. PEG-PDLA stabilized nanoemulsions manifested lower hematological toxicity than corresponding micelles suggesting higher drug retention in circulation. Summarizing, micelles with elastic cores appear preferable to those with solid cores as drug carriers. Micelles with elastic cores and corresponding nanoemulsions both manifest high therapeutic efficacy, with nanoemulsions exerting lower systemic toxicity than micelles. The presence of a small fraction of micelles with elastic cores in nanoemulsion formulations is desirable for prevention of the development of drug resistance. Key words: Polymeric micelles; nanoemulsions; paclitaxel; tumor recurrence; drug resistance; hematological toxicity; poly(ethylene oxide)-co-poly(L-lactide); poly(ethylene oxide)-co-poly(D,Llactide).

ACCEPTED MANUSCRIPT

1. Introduction

AC CE P

TE

D

MA

NU

SC

RI

PT

During the last two decades, polymeric micelles and nanoemulsions have been under extensive development. Micelles are spherical nanoparticles with a core-shell structure having hydrophobic cores and hydrophilic shells; hydrophobic micelle cores serve for encapsulation of lipophilic drugs. Micelle formation is thermodynamically driven; micelles are formed by self-assembly of individual molecules (unimers) of amphiphilic block copolymers. Nanoemulsion droplets are also spherical particles with hydrophobic cores made of oil; nanoemulsions are stabilized with surfactants forming droplet shells; the most widely used nanodroplet stabilizers are phospholipids [1, 2]; micelle-forming block copolymers may be also used for nanodroplet stabilization. In this case, nanodroplet shells have two layers [3-7]; the inner layer is formed by the hydrophobic block of the block copolymer while the outer layer is a hydrophilic block, usually poly(ethylene oxide) (PEG). A lipophilic drug is associated with the inner hydrophobic layer of the shell as illustrated in Figure 1. Nanodroplets sizes (200 nm to 1000 nm) are significantly larger than those of micelles (20 nm to 100 nm).

Figure 1: A schematic representation of the nanodroplet structure. PFC represents perfluorocarbon used as oil.

The most thoroughly studied micellar systems comprise PEG as a hydrophilic block (or blocks) and polyesters or poly(amino acids) as lipophilic blocks. The micelles formed by poly(ethylene oxide)-co-poly(L-lactide) (PEG-PLLA), poly(ethylene oxide)-co-poly(D,L-lactide) (PEGPDLA) or poly(ethylene oxide)-co-poly(caprolactone) (PEG-PCL) are pH-sensitive and biodegradable. If internalized by tumor cells via endocytosis, the micelles end up in the acidic environment of endosomes and lysosomes, where their hydrolysis results in drug release. Polymeric micelles have a number of advantages over other types of drug carriers [5, 8-15]. Drug encapsulation in micelles may dramatically increase aqueous concentrations of bioactive compounds and prevent them from degradation degrading while in circulation. Use of polymeric micelles as solubilizing agents allows for the replacement of highly toxic solvents [16-18]. In addition, micellar encapsulation is expected to provide for tumor targeting through the enhanced permeability and retention effect.

ACCEPTED MANUSCRIPT

RI

PT

Despite advantageous properties and excellent preclinical results, only a few polymeric micelles have progressed to clinical trials and only one type of micelles reached the market [19-21]. In clinical trials, micellar formulations of doxorubicin (DOX) manifested the same spectrum of side effects as free DOX suggesting a lack of effective tumor targeting [22]. For micellar encapsulated paclitaxel (PTX), a rapid loss of drug from micellar carriers was observed. Despite unfavorable pharmacokinetic parameters [17], the PXT-loaded poly(ethylene oxide)-copoly(D,L-lactide) (PEO2000-co-PDLA1750) micelles (Genexol®-PM) showed promising therapeutic results for breast, lung, and pancreatic cancer in clinical trials [19-21].

AC CE P

TE

D

MA

NU

SC

There may be a number of underlying reasons for the current lack of micelle clinical success. The major reason is probably associated with a premature drug release in circulation. To prevent premature degradation, micelles can be strengthened by the introduction of strong hydrophobic interactions into micelles cores. This may be achieved, for instance, by the introduction of oil [23] or mixtures of hydrophobic drugs [24]. Recently, we suggested using for this purpose perfluorocarbon (PFC) compounds to act as the oil [3, 4, 6, 7, 25, 26]. In this approach, drugloaded polymeric micelles serve as starting points for developing drug-loaded nanoemulsions. The phase state of these systems depends on the copolymer/PFC concentration ratio [3]. At low PFC concentrations, the PFC dissolves in micelles cores and stabilizes them. At higher PFC concentrations that exceed PFC solubility limit in micelles cores, PFC evolves in a separate phase as a nanodroplet, and a PFC nanoemulsion stabilized with a block copolymer shell is formed (Figure 1). In some range of the PFC/copolymer concentration ratios, micelles coexist with nanodroplets. When PFC concentration is further increased, all block copolymer is used for the nanodroplet stabilization; micelles disappear and only nanodroplets are present in the formulation. The size of nanoemulsion droplets increases with an increase in PFC/copolymer concentration ratio. Upon dilution, nanodroplets manifested much higher stability than micelles; based on dynamic light scattering measurements, nanodroplets were preserved upon a hundredfold dilution of the initial formulation while micelles were completely destroyed. Both PTX-loaded micelles and nanodroplets manifested strong therapeutic effects in preclinical studies of breast, ovarian, and pancreatic cancer [6, 25, 26]. However the effect of the block copolymer structure and side-by-side comparison of micelles and nanoemulsions for their therapeutic efficacy and systemic toxicity has not been previously performed. In the current study, tumor resolution and recurrence, development of drug resistance, histological evaluation of initial and recurrent tumors, as well as hematological toxicity of PTX-loaded PEG-PDLA micelles and PEG-PDLA stabilized nanoemulsions were compared. 2. Materials and Methods 2.1 Block copolymer. Water soluble, biodegradable block copolymer poly(ethylene oxide)-copoly(D,L-Lactide) (PEG-PDLA) with molecular weight of either block of 2000 Da (Akina, Inc., West Lafayette, IN, USA) was used in this study. 2.2 Micelle preparation. The empty or PTX-loaded PEG-PDLA micellar solutions were prepared by a solid dispersion technique. Typically, 10 or 20 mg PEG-PDLA (with or without 5 mg PTX) were dissolved in 1 ml tetrahydrofuran (THF). The THF was then evaporated under a gentle nitrogen stream at 60 °C or pumped out at room temperature. Micelles were reconstituted by dissolving residual gel matrix in 1 ml phosphate buffered saline (PBS, pH 7.4). The hydrodynamic size of micelles measured by dynamic light scattering was 40 – 60 nm.

ACCEPTED MANUSCRIPT

PT

2.3 Nanodroplet preparation. Perfluoro-15-crown-5-ether (PFCE) (Oakwoods Products, Inc., West Columbia, SC, USA) was introduced at a concentration of 1% to 4% (vol.) into empty or PTX-loaded micellar solution and emulsified by sonication on ice (VCX500, Sonics and Materials, Inc., CT, USA). Micellar solutions and perfluorocarbon compounds were sterilized by filtration and mixed in a sterile test tube before being sonicated. The size of PFCE nanodroplets (both empty and drug loaded) was in the range of 250 nm to 350 nm.

SC

RI

2.4 Drug. Paclitaxel (PTX) was obtained from LC Laboratories (Woburn, MA, USA). Green fluorescence labeled PTX (F-PTX) was obtained from Molecular Probes (Life Technologies, NY).

NU

2.5 Particle size distribution. Size distribution of nanoparticles was measured by dynamic light scattering at a scattering angle of 165° using Delsa Nano S instrument (Beckman Coulter, Osaka, Japan) equipped with a 658-nm laser and a temperature controller. Particle size distribution was analyzed using the non-negative least squares (NNLS) method.

AC CE P

TE

D

MA

2.6 Subcutaneous PDA MiaPaCa-2 tumor model. The experiments were approved by the University of Utah Institutional Animal Care and Use Committee. Human pancreatic cancer MiaPaCa-2 cells were obtained from the American Type Culture Collection (Rockville, MD, USA) and transfected with red fluorescence protein (RFP) [27]. The excitation and emission peaks for the RFP were 563 nm and 587 nm, respectively. Cells were maintained in DMEM media supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, USA) at 37°C in a 5% CO2 incubator. Male nude mice between 6-8 weeks of age were utilized (NCr-Nu/Nu, National Cancer Institute, Frederick, MD, USA). For the tumor induction, mice were anesthetized with isoflurane and received a single subcutaneous injection of 1.5x106 MiaPaCa-2 cells suspended in 150µL of serum free DMEM. Tumors were allowed to progress until reaching a volume of 600 to 700 mm3, at which point mice were randomly assigned to a treatment group. All tumors manifested bright fluorescence. Tumors were measured with calipers twice weekly. Tumor volume was calculated as follows: V = L x W2 / 2

(1)

where L and W are the length and the width of the tumor, respectively. 2.7 Comparison of the therapeutic efficacy of PEG-PDLA micelles and PEG-PDLA stabilized perfluoro-15-crown-5-ether (PFCE) nanodroplets. MiaPaCa-2 pancreatic cancer bearing mice were treated with fifteen 200-µl systemic injections of either a 0.5% PTX/2%PEG-PDLA (micellar) or 0.5% PTX/1% PFCE/2%PEG-PDLA (nanodroplet) formulation given twice weekly for 7.5 weeks. The representative size distribution for the above nanoemulsion is shown in Figure 2. Some fraction of micelles was present in the nanoemulsion formulation. PTX dose used was 40 mg/kg for all the formulations. Treatment started when tumors reached a volume between 400 mm3 and 700 mm3 (tumor size of 8 mm to 10 mm). Mice were randomly divided to two groups, 3 animals per group. 2.8 Blood tests. Healthy mice were randomly divided into four groups, three animals per group, and intravenously injected with 400 µl of one of the following formulation: 1. Phosphate buffer solution (PBS) (control)

ACCEPTED MANUSCRIPT 2. 0.5% PTX/2% PEG-PDLA (micelles) 3. 3% PFCE/2% PEG-PDLA nanodroplets (empty nanoemulsions) 4. 0.5% PTX/3% PFCE/2% PEG-PDLA nanodroplets (PTX-loaded nanoemulsions)

MA

NU

SC

RI

PT

Blood tests were taken 48 hours after the injection. Size distributions of the formulations used in the blood tests are provided in Table1.

TE

D

Figure 2: Size distribution for the composition of 1% PFCE/2%PEG-PDLA/0.5% PTX nanoemulsion used in a therapeutic efficacy studies; mean micelle size was 58.8 nm; mean nanodroplet size was 214.6 nm.

AC CE P

Table 1. Size distributions for formulations used in the therapeutic efficacy studies and blood tests.

Formulation

Micelles size, nm

Nanodroplet size, nm

Micelles, PTX-loaded (blood test and therapeutic efficacy) 2% PEG-PDLA/0.5% PTX

50.8 nm / 100%

Droplets, PTX-loaded (blood tests) 3% PFCE/2% PEG-PDLA/0.5% PTX

-

351 nm / 100%

Droplets, empty (blood tests) 3% PFCE/2% PEG-PDLA

-

296nm /100%

Droplets, PTX-loaded (therapeutic efficacy) 1% PFCE/2%PEG-PDLA/0.5% PTX

58.8

-

214

ACCEPTED MANUSCRIPT

NU

SC

RI

PT

2.9. Comparison of drug retention by micelles and nanodroplets. FluorescentPTX (F-PTX) labeled with Oregon Green (Molecular Probes, Grand Island, NY,USA) was introduced into 2% PEG-PDLA micelles or 4% PFCE/2% PEG-PDLA nanodroplets as described above for the PTX loading at a concentration of 20µg/ml. No micelles remained in the nanodroplet formulation. Formulations were dialyzed through a 2.5 kDa cellulose membrane (Life Technologies, Grand Island, NY, USA) 2.10 Histological evaluations. Histological examination was performed for two controlled tumors and two recurrent tumors as described in the Results section. Segments of tumor were fixed in 10% formalin, dehydrated, embedded in paraffin, sectioned (5m), and stained with haematoxylin and eosin (H&E). Histological evaluation was performed in two centers with the help of Drs. Natalia Vykhodtseva and Natan McDannold (Brigham and Women’s Hospital, Harvard Medical School) and Anna Shurlygina (Institute of Physiology and Fundamental Medicine, Siberian Branch of the Russian Academy of Medical Sciences).

MA

3. Results

AC CE P

TE

D

3.1 Comparison of the therapeutic efficacy of PTX-loaded micelles and nanodroplets Animals were treated with 15 injections of either 2% PEG-PDLA/0.5% PTX micellar or 1% PFCE/2%PEG-PDLA/0.5% PTX nanoemulsion formulation given twice weekly for 7.5 weeks without the application of ultrasound. Strong therapeutic effect with complete tumor resolution was observed for both, micelles and nanoemulsions (Figure 3A). For micelles, tumors appeared completely resolved in less than 3 weeks after the start of the treatment. For the nanoemulsions, complete tumor resolution proceeded slightly slower; tumor regression continued after the end of the treatment until tumors were completely resolved. While the survival rate of control animals was about 3 weeks, all treated mice survived for at least 6 months upon which they were sacrificed. Survival data for control and treated mice are presented in Table 2. Table 2. Average life span of animals treated with PTX-loaded PEG-PDLA formulations with and without application of tumor-directed focused ultrasound (MRgFUS). N 3 7 8 4 3 3

Treatment group Negative control PTX- nanoemulsion, no ultrasound (single treatment) PTX-nanoemulsion + ultrasound (single treatment PTX-nanoemulsion, no ultrasound (15 treatments) PTX-micelles, no ultrasound (15 treatments)

Life span (week) 3.5± 0.5 7.0± 1.0 10.3±1.6* Long-term survivors Long-term survivors Long-term survivors

ACCEPTED MANUSCRIPT

MA

NU

SC

RI

PT

A

D

B

TE

1 0.8 0.6

AC CE P

normalized tumor volume

1.2

micelles

0.4

nanodroplets

0.2

0

0

50

100

days after first treatment

Figure 3. A - Time courses of tumor regression in pancreatic cancer bearing mice treated with 15 injections of PTX (40 mg/kg) encapsulated in either 2%PEG-PDLA micelles (N=3) or 1% PFCE/2% PEG-PDLA Nanoemulsions (N=3) given twice weekly for 7.5 weeks. Left arrow indicates the end of the first treatment; right arrow indicates the start of the second treatment of a recurrent tumor. Survival rate of control animals was about 3 weeks; all treated mice survived for at least 6 months upon which they were sacrificed. B - Average tumor regression curves for the data presented in Figure 3A. Mean values plus/minus standard deviations are presented up to the time of the onset of the recurrence of one of the tumors; N=3 for each treatment protocol. Though tumor regression appeared slightly slower for the nanoemulsion treated mice, all six tumors treated with either micelles or nanoemulsions were completely resolved. The small cohort of animals precluded rigorous statistical treatment of differences between micelles and nanoemulsions; based on the current data, differences were not statistically significant.

ACCEPTED MANUSCRIPT

SC

RI

PT

3.2 Tumor recurrence In one of the three animals treated with a micellar PTX composition, the tumor recurred at the initial site about 12 weeks after the complete resolution of the initial tumor (14 weeks after the start of the treatment). The recurrent tumor was not fluorescent, indicating its growth from a genetically different cell (possible mechanism of this unexpected effect is beyond a scope of the current paper). The recurrent tumor grew much slower than control untreated tumors (Figure 3). When the recurrent tumor reached the same size as the initial tumor (V = 620 mm 3), it was retreated using the initial protocol (i.e. micellar-encapsulated PTX injected twice weekly for 4 weeks, for a total of 8 injections). The tumor manifested sensitivity to the second therapy; when the residual volume of the recurrent tumor dropped to about 55 mm3, the animal was sacrificed; tumor was extracted, sliced and stained with Hematoxilin/eosin for the histological examination.

AC CE P

TE

D

MA

NU

No tumor recurrence was observed in animals treated with fifteen injections of PTX-loaded nanoemulsions. However, tumor recurrence was observed in one of four animals treated with a single injection of PTX-loaded nanoemulsion combined with the application of focused ultrasound under the MRI control (MRgFUS) according to the protocol described in ref. [6] (Figure 4). The resolution of this tumor occurred as fast as that of the tumors treated with multiple injections of micellar encapsulated PTX without ultrasound (Figure 4); however, tumor recurrence occurred much faster (in 3 weeks rather than 12 weeks after the tumor resolution). The recurrent tumor retained fluorescence and grew almost as fast as control tumors (Figure 4); the recurrent tumor was allowed to grow to the size of 556 mm3, after which it was treated with nine injections of PTX-loaded nanoemulsion given twice weekly without ultrasound. Note that both recurrent tumors were of close size at the start of the second treatment; their treatment protocols (multiple injections without ultrasound) differed only in the type of the drug nanocarrier (micelles vs. nanoemulsions). For both formulations, recurrent tumors manifested sensitivity to the second treatment indicating that in the course of the first treatment, drug resistance has not been developed.

normalized tumor size

6 5 4 3

single injection, US 15 injections, no US

2

control 1 0 0

50

100

150

200

time after first treatment, days Figure 4: Tumor regression, regrowth, and the effect of the second treatment. Open triangles – untreated control. Diamonds – the primary tumor (initial size 164 mm3) was treated with a single injection of a

ACCEPTED MANUSCRIPT PTX-loaded nanoemulsion combined with the application of focused ultrasound under the MRI control (MRgFUS); ultrasound at sub-ablative power level of 3.1 W was applied 8 h after the injection; only a fraction of the tumor was irradiated. The tumor had been completely resolved but the regrowth started 6 weeks after the treatment. When the recurred tumor reached the volume of about 600 mm3, it was treated with nine injections of the nanoemulsion formulation given twice weekly without ultrasound.

PT

When the volume of the recurrent tumor dropped to 112 mm3, the animal was sacrificed, tumor was extracted, sliced and stained with Hematoxilin/eosin for the histological examination.

SC

RI

Squares – the primary tumor (initial volume 630 mm3) was treated with fifteen injections of PTX-loaded micelles; the tumor had been completely resolved but the regrowth started 14 weeks after the start of the first treatment. The recurrent tumor was treated with eight injections of the micellar PTX formulation; when the tumor volume dropped to 55 mm3, the mouse was sacrificed for the histological evaluation of the residual tumor. Arrows indicate onset of the corresponding second treatment. US - ultrasound.

AC CE P

TE

D

MA

NU

3.3 Histological examination of recurred tumors The detailed description of histological results is presented in Figures S1 A-C of Supplemental material. A histological section of the interior region of a large control untreated tumor (V = 2,300 mm3) is shown in Figure 5A. A well-defined pronounced stroma, 10 mitotic cells, 24 macrophages, some necrosis, and mononuclear leucocyte infiltration characterize the control tumor. Histological sections of the recurrent tumor treated with PTX-loaded micelles are presented in Figure 5B. The primary tumor was treated with 15 injections of PTX-loaded micelles; the recurrent tumor was treated with 8 injections of the same formulation. In the left panel, there is a region of a connective tissue (fibroblasts) imbedded between tumor cells, which characterizes the onset of the scar formation. Tumor regions are characterized by significant pericellular and interstitial swelling and formation of cysts. Only one mitotic cell is observed in the field of view. Stroma is not significant. The right panel shows the area of a swelled scar tissue with a pronounced hemorrhage; tumor cells are absent indicating efficient therapy of the recurrent tumor with micellar-encapsulated PTX. Histological sections of the recurrent tumor treated with the injections of PTX-loaded nanodroplets are shown in Figure 5C. The primary tumor was treated with a single injection of PTX-loaded nanodroplets combined with the application of MRgFUS. The recurrent tumor was treated with 9 injections of PTX-loaded nanodroplets without ultrasound. In the left panel, there is an island of degenerated and necrotic tumor tissue with significant pericellular swelling; 3 mitotic cells are still observed. Necrotic masses are visible between the cells, with the growing collagen fibrils and capillaries characterizing the onset of the scar formation. A swollen connective tissue is infiltrated with leucocytes and contains capillaries and small blood vessels. In the right panel, small islands of degenerated and necrotic tumor cells are embedded into the connective tissue; no mitoses are observed. The tissue is swollen, with the formation of microcysts and distinct areas of hemorrhage. Stroma is not significant. The connective tissue is represented by bundles of rough filaments with enlarged capillaries. Lymphatic vessels are seen in this slide; no tumor cells are observed in their lumens, which may indicate low metastatic activity; however, more rigorous statistical treatment would be needed to support the last conclusion. Again, histological examination indicates effective therapy of the recurrent tumor with nanoemulsion encapsulated PTX.

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 5: Histological sections of (A) the interior region of a large control untreated tumor; (B) the recurrent tumor treated with the injections of PTX-loaded micelles; (C) the recurrent tumor treated with the injections of PTX-loaded nanodroplets.

In conclusion, cell proliferation and stroma expression were significantly decreased in the treated tumors in comparison to control; the regions of hemorrhage were more pronounced in the tumor treated with the micellar-encapsulated PTX in comparison with nanodroplet-encapsulated PTX. Tumor tissue infiltration with macrophages and neutrophils was significantly decreased in the nanodroplet treated tumor in comparison with the micellar treated tumor and the control nontreated tumor. However in the surrounding connective tissue, the macrophage and neutrophil infiltration was higher for the nanodroplet treatment. Necrosis was more pronounced in the interior of a large control tumor in comparison to the smaller residual tumors of treated animals. The qualitative indices of histological sections are presented in Table 3. Table 3. The qualitative indices of histological sections. Parameter

Control

Micellar encapsulated Nanodroplet PTX encapsulated PTX

ACCEPTED MANUSCRIPT ++

+

-

Swelling of tumor tissue/connective tissue

++/- (there was no connective tissue)

++/+

++/++

Hemorrhage of the tumor/connective tissue

+/-( there was no connective tissue)

++/++

Necrosis

++

+

PT

Stroma

SC

RI

+/+ +

TE

D

MA

NU

3.4 Comparison of the hematological toxicity of PTX encapsulated in micelles or nanodroplets In these experiments, blood tests of mice injected with the same PTX dose of 80 mg/kg encapsulated in either micelles or nanodroplets were performed; perfluorocarbon concentration in the nanodroplet formulation was increased to 3% to ensure the absence of micelles [3, 4]. Four groups of animals were tested. Mice were intravenously injected with 400 l of one of the following formulations: PBS (control); 0.5% PTX/2% PEG-PDLA (micelles); 0.5% PTX/2% PEG-PDLA/3% PFCE (nanodroplets); empty nanodroplets (2% PEG-PDLA/3% PFCE) were also tested for non-drug related toxicity. Blood tests were performed 48 hours after the injection. The results are presented in Figure 6A-E.

AC CE P

The hematological toxicity was significantly higher for the micelle-encapsulated PTX in comparison to the nanodroplet-encapsulated PTX. No hematological toxicity of empty nanodroplets was observed. Note that in these experiments, the copolymer and perfluorocarbon doses were correspondingly 2-fold and 6-fold higher than their doses in preclinical studies. The majority of drug toxicity was exerted on the white blood cells. These data indicate that the toxicities reported above were drug-related. A very pronounced hematological toxicity (neutropenia etc.) of a micelle-encapsulated PTX suggested a lack of the micelle protection action. This may be associated with micelle dissociation in circulation resulting in a premature release of the encapsulated PTX, which increases systemic toxicity of the drug. The nanodroplets appeared to retain the drug more effectively than micelles, resulting in a decrease in systemic drug toxicity. Stronger retention of drug by nanodroplets than for micelles was confirmed in a dialysis study using fluorescently labeled PTX (F-PTX) as a model drug as described in the Methods section (Figure 7).

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Figure 6: The effect of various PTX formulations and empty nanodroplets on the blood cell counts; A – Red blood cells; B – white blood cells; C – lymphocytes; D – neutrophils; and E – platelets.

ACCEPTED MANUSCRIPT

PT

2000000

RI

1500000

SC

Micelles Nanodroplets

NU

1000000

500000

0 0

100

200

MA

Fluorescence intensity (a.u.)

2500000

300

400

500

TE

D

Time (min)

4. Discussion

AC CE P

Figure 7. Drug (F-PTX) release from micelles (diamonds) or nanodroplets (squares) as measured by dialysis through a 2.5 kDa membrane (see Methods). Micelle composition: 2% PEG-PDLA; nanodroplet composition: 4% PFCE/2% PEG-PDLA. Average micelle size 50 ± 5 nm; nanodroplet size 330 ± 30 nm.

4.1 Thermodynamic and kinetic stability of micelles Block copolymer micelles are formed by a self-assembly of amphiphilic block copolymer molecules in aqueous milieu. The self-assembly occurs when copolymer concentration exceeds some critical value called critical micelle concentration (CMC). Below the CMC, block copolymer molecules exist in a solution in the form of individual molecules (unimers); therefore when copolymer concentration drops below the CMC, micelles dissociate into unimers. Because systemic injections of micellar formulations are associated with very substantial dilutions in the circulatory system, micelles may dissociate into unimers upon injection. When micelle dissociates, poorly soluble drugs may either stay associated with hydrophobic blocks of unimers, get associated with biological carriers (e.g. albumin), or, at higher concentration, precipitate inside blood vessels. To prevent a premature micelle dissociation, micellar systems should either have low CMC (i.e. manifest thermodynamic stability), or have slowly dissociating cores (i.e. manifest kinetic stability). Kinetic stability of micelles depends on a physical state of micelle cores and is the highest for micelles with solid cores such as those formed by PLLA blocks. PLLA glass transition temperature is above physiological temperature (Tg = 60-65 ºC) thus PLLA remains in a glassy state in vivo; in addition, a regular structure of PLLA blocks favors crystallization. Micelles such

ACCEPTED MANUSCRIPT as PEG-PDLA or PEG-PCL have elastic cores (for PCL, Tg = – 60 ºC; for PDLA, Tg is below physiological temperature).

RI

PT

The high strength of micelles with solid cores has its downside as it may result in excessively tight drug retention, slow the rate of micelle biodegradation, and thus not provide satisfactory kinetics of drug release. Therefore micelles with solid cores may require external stimulation to release drugs [8].

MA

NU

SC

Micelles with elastic cores are in equilibrium with their unimers; the equilibrium concentration of the latter is equal to the CMC. If unimers are eliminated from a micellar solution (e.g. via dialysis), micelles dissociate into unimers to restore the equilibrium. We have recently reported fast extravasation of PEG-PDLA unimers observed in vivo using the intravital fluorescence imaging [28]. We have suggested that this mechanism may be responsible for dissociation of micelle with elastic cores in the circulatory system even if micelles are thermodynamically stable and their concentration in the vasculature remains above the CMC. However, the weakness of micelle with elastic cores has a very important advantage of preventing the development of drug resistance, which is attributed to the action of copolymer unimers [29-32]. In addition, water and acids diffuse faster into elastic cores, which accelerate the biodegradation and drug release.

AC CE P

TE

D

4.2 Therapeutic efficacy of micelles and nanoemulsions: effect of the structure of a hydrophobic block of a block copolymer Comparison of the data presented above and in refs. [6, 26] for the PEG-PDLA stabilized formulations with those reported earlier for those stabilized with PEG-PLLA [25] indicated that stability and therapeutic efficacy of micelles and nanoemulsions depends strongly on the type and structure of a hydrophobic block of a block copolymer. The PEG-PLLA block copolymer used in the first generation of our formulations formed micelles with solid cores and nanodroplets with solid inner layer of the shells [25, 33]. Ultrasound imaging indicated that PEG-PLLA-stabilized nanodroplets were extremely stable; when PEG-PLLA-stabilized nanodroplets were injected directly into a mouse tumor or a porcine thigh muscle, they remained at the site of injection for many weeks or even months without any signs of biodegradation (data not shown). These nanodroplets could be considered to act as nanoimplants. The drug (PTX) was tightly retained in the nanodroplets and ultrasound stimulation was required to trigger the release of the encapsulated drug and induce therapeutic effect [25]. In the second generation of nanoemulsions, PEG-PDLA copolymer with the elastic PDLA block was used as a nanodroplet stabilizer [6, 26, 34]. Ultrasound imaging showed that these nanodroplets disappeared from the site of injection within two days, presumable due to a fast PEG-PDLA biodegradation. In accordance with that, the PTX-loaded PEG-PDLA stabilized nanoemulsions exerted a strong therapeutic effect (i.e. dramatically extended life span, suppression of metastases and ascites in pancreatic tumor bearing mice) even without ultrasound application. However, ultrasound enhanced the therapeutic effect [6, 26]. High therapeutic efficacy of PTX-loaded PEG-PDLA stabilized micelles and nanoemulsions suggested that micelles and nanodroplets successfully accumulated in the tumor tissue and effectively delivered drug to tumor cells. Intravital fluorescence imaging showed that the extravasation and internalization of PEG-PDLA micelles was fast, while that of nanodroplets was significantly slower (slow internalization of nanodroplets is illustrated in Figure S2 of

ACCEPTED MANUSCRIPT Supplement material) [28]. We hypothesize that the high therapeutic efficacy of PTX-loaded PEG-PDLA nanoemulsions is associated with fast in vivo biodegradation of the PEG-PDLA copolymer resulting in droplet degradation and drug release.

SC

RI

PT

4.3 Suppression of the development of drug resistance Note that the recurrent tumors treated initially with either PTX/PEG-PDLA micelles or PTX/PFCE/PEG-PDLA nanodroplets did not develop drug resistance. This is a dramatic improvement in therapeutic outcome in comparison to the treatment with PTX/PEG-PLLA micelles or nanodroplets, where drug resistance developed in the course of the first treatment and repeated treatments with the same protocol appeared ineffective [25].

NU

As mentioned above, the prevention of drug resistance has been attributed to the action of copolymer unimers [29-32]. Due to their high kinetic stability, micelles with solid PLLA cores do not release unimers, which may account for the development of drug resistance when PEGPLLA was used [25].

AC CE P

TE

D

MA

We hypothesize that the prevention of the development of drug resistance at treatments with PEG-PDLA stabilized formulations was associated with the presence of copolymer unimers in both, micellar and nanodroplet formulations. The nanoemulsion composition used here contained a small fraction of micelles (Figure 2), which proved to be sufficient for preventing the development of drug resistance. This is of paramount importance for further clinical applications of drug-loaded nanoemulsions that should presumably contain a small fraction of micelles with elastic cores; these may be residual micelles from which the nanodroplets were manufactured or other polymeric micelles with elastic cores. Note that PEG-PDLA stabilized nanoemulsion formulations that did not contain micelles (1% PFCE/5% PDLA) did not prevent the development of drug resistance [26]. We want to underline that less than 10% (vol.) of micelles in the nanoemulsion formulation was sufficient for preventing drug resistance; this is important because PTX-loaded micelles manifested higher hematological toxicity than nanoemulsions. It is noteworthy that the initial tumor regression rates were similar for the animals treated with a single nanodroplet injection combined with ultrasound or multiple injections without ultrasound. In contrast, the recurrence of the resolved tumor occurred much faster in the animal treated with a single nanodroplet/ultrasound injection and the tumor also grew at a faster rate (Figure 4). For nanoemulsions stabilized with copolymers with elastic cores that can be used without ultrasound, multiple injections without ultrasound may be preferred to a single injection with the application of MRgFUS.

5.

Conclusions

Micelles and nanoemulsions formed with PEG-PDLA block copolymer manifested a number of important advantages in the therapeutic efficacy and prevention of drug resistance over corresponding formulations formed with PEG-PLLA copolymer. This was attributed to the elastic physical state of micelle cores and nanodroplet shells that allowed release of unimers and

ACCEPTED MANUSCRIPT

SC

RI

PT

provided an adequate biodegradation rate. PTX-loaded PEG-PDLA nanoemulsions manifested lower hematological toxicity than corresponding micelles, which makes them more attractive drug carriers; however a small fraction of micelles should be present in the nanoemulsion formulation for the prevention of the development of drug resistance. Summarizing, micelles with elastic cores appear preferable to those with solid cores as drug carriers. Micelles with elastic cores and corresponding nanoemulsions both manifest high therapeutic efficacy, with nanoemulsions exerting lower systemic toxicity than micelles. The presence of a small fraction of micelles with elastic cores in nanoemulsion formulations is desirable for prevention of the development of drug resistance.

AC CE P

TE

D

MA

NU

Acknowledgment: The work was supported by the NIH R01 EB 001033 grant to NR.

ACCEPTED MANUSCRIPT References

AC CE P

TE

D

MA

NU

SC

RI

PT

[1] P.M. Winter, K. Cai, S.D. Caruthers, S.A. Wickline, G.M. Lanza, Emerging nanomedicine opportunities with perfluorocarbon nanoparticles, Expert Rev Med Devices, 4 (2007) 137-145. [2] H.F. Zhou, H.W. Chan, S.A. Wickline, G.M. Lanza, C.T. Pham, Alphavbeta3-targeted nanotherapy suppresses inflammatory arthritis in mice, FASEB J, 23 (2009) 2978-2985. [3] Z. Gao, A.M. Kennedy, D.A. Christensen, N.Y. Rapoport, Drug-loaded nano/microbubbles for combining ultrasonography and targeted chemotherapy, Ultrasonics, 48 (2008) 260-270. [4] N. Rapoport, Phase-shift, stimuli-responsive perfluorocarbon nanodroplets for drug delivery to cancer, Wiley Interdiscip Rev Nanomed Nanobiotechnol, 4 (2012) 492-510. [5] N. Rapoport, Ultrasound-mediated micellar drug delivery, Int J Hyperthermia, 28 (2012) 374-385. [6] N. Rapoport, A. Payne, C. Dillon, J. Shea, A.R. Scaife, R. Gupta, Focused Ultrasound-Mediated Drug Delivery to Pancreatic Cancer in a Mouse Model, Journal of Therapeutic Ultrasound, 1 (2013). [7] N. Rapoport, Z. Gao, A. Kennedy, Multifunctional nanoparticles for combining ultrasonic tumor imaging and targeted chemotherapy, J Natl Cancer Inst, 99 (2007) 1095-1106. [8] N. Rapoport, Physical stimuli-responsive polymeric micelles for anti-cancer drug delivery, Prog Polym Sci, 32 (2007) 962-990. [9] R.R. Sawant, V.P. Torchilin, Polymeric micelles: polyethylene glycol-phosphatidylethanolamine (PEGPE)-based micelles as an example, Methods Mol Biol, 624 (2010) 131-149. [10] V.P. Torchilin, Drug targeting, Eur J Pharm Sci, 11 Suppl 2 (2000) S81-91. [11] V.P. Torchilin, Targeted polymeric micelles for delivery of poorly soluble drugs, Cell Mol Life Sci, 61 (2004) 2549-2559. [12] Y. Kakizawa, K. Kataoka, Block copolymer micelles for delivery of gene and related compounds, Advanced drug delivery reviews, 54 (2002) 203-222. [13] K. Kataoka, A. Harada, Y. Nagasaki, Block copolymer micelles for drug delivery: design, characterization and biological significance, Advanced drug delivery reviews, 47 (2001) 113-131. [14] Y. Matsumura, M. Yokoyama, K. Kataoka, T. Okano, Y. Sakurai, T. Kawaguchi, T. Kakizoe, Reduction of the side effects of an antitumor agent, KRN5500, by incorporation of the drug into polymeric micelles, Jpn J Cancer Res, 90 (1999) 122-128. [15] Y. Yamamoto, Y. Nagasaki, Y. Kato, Y. Sugiyama, K. Kataoka, Long-circulating poly(ethylene glycol)poly(D,L-lactide) block copolymer micelles with modulated surface charge, J Control Release, 77 (2001) 27-38. [16] E. Fournier, M.H. Dufresne, D.C. Smith, M. Ranger, J.C. Leroux, A novel one-step drug-loading procedure for water-soluble amphiphilic nanocarriers, Pharm Res, 21 (2004) 962-968. [17] T.Y. Kim, D.W. Kim, J.Y. Chung, S.G. Shin, S.C. Kim, D.S. Heo, N.K. Kim, Y.J. Bang, Phase I and pharmacokinetic study of Genexol-PM, a cremophor-free, polymeric micelle-formulated paclitaxel, in patients with advanced malignancies, Clin Cancer Res, 10 (2004) 3708-3716. [18] D. Le Garrec, S. Gori, L. Luo, D. Lessard, D. Smith, M.e.a. Yessine, Poly(n-vinylpyrrolidone)-blockpoly(d,l-lactide) as a new polymeric solubilizer for hydrophobic anticancer drugs: In vitro and in vivo evaluation, J Control Release 99 (2004) 83-101. [19] J.G. Kim, S.K. Sohn, H.S. Song, K.Y. Kwon, Y.R. Do, K.H. Lee, M.S. Hyun, H.M. Ryoo, S.H. Bae, K.U. Park, J.H. Baek, W.S. Lee, J.S. Chung, G.J. Cho, C.H. Sohn, J.S. Jang, H.Y. Chung, W. Yu, Multicenter phase II study of weekly paclitaxel plus cisplatin combination chemotherapy in patients with advanced gastric cancer, Cancer Chemother Pharmacol, 60 (2007) 863-869. [20] K.S. Lee, H.C. Chung, S.A. Im, Y.H. Park, C.S. Kim, S.B. Kim, S.Y. Rha, M.Y. Lee, J. Ro, Multicenter phase II trial of Genexol-PM, a Cremophor-free, polymeric micelle formulation of paclitaxel, in patients with metastatic breast cancer, Breast Cancer Res Treat, 108 (2008) 241-250.

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

[21] M.W. Saif, N.A. Podoltsev, M.S. Rubin, J.A. Figueroa, M.Y. Lee, J. Kwon, E. Rowen, J. Yu, R.O. Kerr, Phase II clinical trial of paclitaxel loaded polymeric micelle in patients with advanced pancreatic cancer, Cancer Invest, 28 (2010) 186-194. [22] Y. Matsumura, T. Hamaguchi, T. Ura, K. Muro, Y. Yamada, Y. Shimada, K. Shirao, T. Okusaka, H. Ueno, M. Ikeda, N. Watanabe, Phase I clinical trial and pharmacokinetic evaluation of NK911, a micelleencapsulated doxorubicin, Br J Cancer, 91 (2004) 1775-1781. [23] N. Rapoport, Stabilization and acoustic activation of Pluronic micelles for tumor-targeted drug delivery Colloids and Surface B: Biointerfaces, 3 (1999) 93-111. [24] H.C. Shin, H. Cho, T.C. Lai, K.R. Kozak, J.M. Kolesar, G.S. Kwon, Pharmacokinetic study of 3-in-1 poly(ethylene glycol)-block-poly(D, L-lactic acid) micelles carrying paclitaxel, 17-allylamino-17demethoxygeldanamycin, and rapamycin, J Control Release, 163 (2012) 93-99. [25] N.Y. Rapoport, A.M. Kennedy, J.E. Shea, C.L. Scaife, K.-H. Nam, Controlled and targeted tumor chemotherapy by ultrasound-activated nanoemulsions/microbubbles, J Control Release, 138 (2009) 268276. [26] N. Rapoport, K.-H. Nam, R. Gupta, Z. Gao, P. Mohan, A. Payne, N. Todd, X. Liu, T. Kim, J. Shea, C. Scaife, A.M. Kennedy, D.L. Parker, E.-K. Jeong, Ultrasound-mediated tumor imaging and nanotherapy using drug loaded, block copolymer stabilized perfluorocarbon nanoemulsions., J. Control Release, 153 (2011) 4-15. [27] M.J. Torgenson, J.E. Shea, M.A. Firpo, Q. Dai, S.J. Mulvihill, C.L. Scaife, Natural history of pancreatic cancer recurrence following "curative" resection in athymic mice, J Surg Res, 149 (2008) 57-61. [28] N. Rapoport, R. Gupta, Y.S. Kim, B.E. O'Neill, Polymeric micelles and nanoemulsions as tumortargeted drug carriers: Insight through intravital imaging, J Control Release, 206 (2015) 153-160. [29] D.Y. Alakhova, N.Y. Rapoport, E.V. Batrakova, A.A. Timoshin, S. Li, D. Nicholls, V.Y. Alakhov, A.V. Kabanov, Differential metabolic responses to pluronic in MDR and non-MDR cells: a novel pathway for chemosensitization of drug resistant cancers, J Control Release, 142 (2010) 89-100. [30] E.V. Batrakova, A.V. Kabanov, Pluronic block copolymers: evolution of drug delivery concept from inert nanocarriers to biological response modifiers, J Control Release, 130 (2008) 98-106. [31] E.V. Batrakova, S. Li, V.Y. Alakhov, W.F. Elmquist, D.W. Miller, A.V. Kabanov, Sensitization of cells overexpressing multidrug-resistant proteins by pluronic P85, Pharm Res, 20 (2003) 1581-1590. [32] E.V. Batrakova, S. Li, A.M. Brynskikh, A.K. Sharma, Y. Li, M. Boska, N. Gong, R.L. Mosley, V.Y. Alakhov, H.E. Gendelman, A.V. Kabanov, Effects of pluronic and doxorubicin on drug uptake, cellular metabolism, apoptosis and tumor inhibition in animal models of MDR cancers, J Control Release, 143 (2010) 290-301. [33] N.Y. Rapoport, A.L. Efros, D.A. Christensen, A.M. Kennedy, K.H. Nam, Microbubble generation in phase-shift nanoemulsions used as anticancer drug carriers, Bub Sci Eng Tech, 1 (2009) 31-39. [34] B.E. O'Neill, N. Rapoport, Phase-shift, stimuli-responsive drug carriers for targeted delivery, Ther Deliv, 2 (2011) 1165-1187.

SC

RI

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

Graphical abstract