Paclitaxel-loaded folate-coated long circulating and pH-sensitive liposomes as a potential drug delivery system: A biodistribution study

Abstract

A range of antitumor agents for cancer treatment is available; however, they show low specificity, which often limit their use. Recently, we have reported the preparation of folate-coated long-circulating and pH-sensitive liposomes (SpHL-folate-PTX) loaded with paclitaxel (PTX), an effective drug for the treatment of solid tumors, including breast cancer. The purpose of this study was to prepare and characterize SpHL-PTX and SpHL-folate-PTX radiolabeled with technetium–99 m (^99mTc). Biodistribution studies and scintigraphic images were performed after intravenous administration of ^99mTc-PTX, ^99mTc-SpHL-PTX and ^99mTc-SpHL-folate-PTX into healthy and tumor-bearing mice. High radiochemical purity (> 98%) and in vitro stability (> 90%) were achieved for both liposome formulations. The pharmacokinetic properties of ^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-folate-DTPA-PTX decreased in a monophasic manner showing half-life of 400.1 and 541.8 min, respectively. Scintigraphic images and biodistribution studies showed a significant uptake in liver, spleen and kidneys, demonstrating these routes as way for excretion. At 8 h post-injection, the liposomal tumor uptake was higher than ^99mTc-PTX. Interesting, 4 h after administration, the liposome folate coated showed higher tumor-to-muscle ratio than ^99mTc-SpHL-DTPA-PTX and ^99mTc-PTX. In conclusion, the liposomal systems, showed high tumor uptake by scintigraphic images, especially the ^99mTc-SpHL-folate-DTPA-PTX that showed a sustained and higher tumor-to-muscle ratio than non-functionalized liposome, which indicate its feasibility as a PTX delivery system to folate positive tumors.

1. Introduction

Cancer is a public health worldwide problem, due to its high prevalence and mortality. A range of antineoplastic agents is available for clinical applications; however, their low specificity leads to toxic effects in healthy tissues, which, in many cases, could limit their use [1–5]. Among the chemotherapeutic agents, paclitaxel (PTX) is one of the most effective and potent drugs used in the treatment of several solid tumors including, breast, ovarian, non-small cell lung cancer, head and neck tumors [3,6–8]. PTX is poorly soluble in water, therefore, it is commercially available as a micellar dispersion in Cremophor EL^® (polyethoxylated castor oil) and dehydrated ethanol (1:1 v/v), which allows its administration by the intravenous route. Nevertheless, several drawbacks have been related to their clinical application, such as hypersensitivity reactions, peripheral sensory neuropathy, and myelo-suppression, besides development of drug resistance [3,6,9–12]. To overcome these problems, liposomes have emerged as an interesting platform for effective drug delivery since they are biocompatible, bio-degradable and nontoxic nanosystems. In addition, encapsulation into liposomes might prevent drug degradation, improve drug solubility and reduce drug distribution to undesired tissues [3,6,13,14]. Nowadays, some formulations with PTX into different nanosystems have been reported and approved for clinical applications. Among them, a conventional liposomal preparation made up of phospatidylcholine and phosphatidylglycerol (in a 9:1 molar ratio, respectively) containing PTX, Lipusu^®, was approved in China, in 2006 for the treatment of ovarian, breast, head and neck cancer, gastric and non-small cell lung carcinoma [3,6,15–17]. In South Korea, a polymeric micelle formulation, Genexol-PM^®, another PTX-nanosystem, has been approved for breast cancer treatment, and, in 2005 FDA approved the Abraxane^®, a PTX albumin-bound nanoparticle formulation, for the treatment of metastatic cancer [18–22]. In general, these nanosystems demonstrated in vivo efficacy similar to Taxol^®, however some studies have demonstrated their similar toxicity as well low selectivity to tumor tissue, after intravenous injection [11,15–22]. In other to overcome this drawback, we reported the preparation of PTX-loaded folate-coated long circulating and pH-sensitive liposomes (SpHL-folate-PTX). The liposomal formulation is composed by dioleylphosphatidylethanolamine (DOPE), cholesteryl hemisuccinate (CHEMS), distearoylphosphatidylethanolamine-polyethylene glycol₂₀₀₀ (DSPE-PEG₂₀₀₀) and distearoylphosphatidyl-ethanolaminepolyethyleneglycol₂₀₀₀-folate (DSPE-PEG₂₀₀₀-folate) in the molar ratio of 5.7:3.8:0.45:0.05, respectively [3]. Previous studies using small angle X-ray diffraction clearly demonstrated the pH-sensitive of SpHL-PTX showing that the presence of CHEMS led to the stabilization of DOPE molecules in a lamellar structure at pH 7.4. Nonetheless, at lower pH, i.e. at tumor tissues, CHEMS is protonated leading to a hexagonal phase, which is essential to release the drug from liposomes [14]. SpHL-PTX revealed a high level of PTX leakage when in contact with an acid medium. Significant difference in PTX leakage was obtained from pH 6.8, and a release around 30% higher were observed. At pH 5.0 a release next to 70.0% were achieved which confirms the pH-sensitivity of the system. In vitro studies on MDA-MB-231 cells, a human breast tumor line, showed a higher cytotoxic activity for liposomal formulation in comparison to the free drug. Worth mentioning was the improved cytotoxicity of folate-coated formulation which suggests a higher uptake of the vesicles explained by superexpression of folate receptors in this cell line [3]. Due to these promising results, it is essential to evaluate the biodistribution profile of these nanoparticles in order to determine the real potential of these formulations as PTX delivery systems. As such, the purpose of this study was to prepare and characterize SpHL-PTX and SpHL-folate-PTX, radiolabeled with technetium–99 m (^99mTc). Biodistribution studies and scintigraphic images were performed after intravenous administration of ^99mTc-PTX, ^99mTc-SpHL-PTX and ^99mTc-SpHL-folate-PTX into healthy and tumor-bearing mice.

2. Materials and methods

2.1. Materials

Paclitaxel was supplied by Quiral Quimica do Brasil S.A (Juiz de Fora, Brazil). Cremophor EL^® and SnCl₂·2H₂O were purchased from Sigma-Aldrich (São Paulo, Brazil). Dioleoylphosphatidylethanolamine (DOPE) and distearoylphosphatidyl-ethanolaminepolyethyleneglycol₂₀₀₀ (DSPE-PEG₂₀₀₀) were acquired from Lipoid GmbH (Ludwigshafen, Germany). Cholesteryl hemisuccinate (CHEMS) was supplied from Sigma Chemical Company (St. Louis, USA). Sodium chloride (NaCl) was obtained from Merck (Rio de Janeiro, Brazil). Acetonitrile HPLC grade was purchased from Fischer Scientific (New Jersey, USA).^99mTc was obtained from an alumina-based ⁹⁹Mo/^99mTc generator. Water was purified using a Milli-Q apparatus (Millipore, Billerica, USA). All other chemicals and reagents used in this study were of analytical grade. MDA-MB-231 (human breast adenocarcinoma) cell line was purchased from American Type Culture Collection (ATCC^® HTB-26™) (Manassas, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, penicillin and streptomycin were supplied by Gibco Life Technologies (Carlsbad, USA). Trypsin-EDTA solution (0.5%) and trypan blue were purchased from Sigma-Aldrich (São Paulo, Brazil). Matrigel was acquired from BD Biosciences (Bedford, MA). Female BALB/c mice (6–8-week-old) were obtained from CEBIO-UFMG (Belo Horizonte, Brazil) and BALB/c nude mice (6–8-week-old) were supplied from IPEN-SP (São Paulo, Brazil). All animal studies were approved by the local Ethics Committee for Animal Experiments (CEUA/UFMG) under the protocol number 409/2013.

2.2. Synthesis of distearoylphosphatidyl-ethanolaminepolyethyleneglycol₂₀₀₀-Diethylene-triaminepentaacetic acid (DSPE-PEG₂₀₀₀-DTPA)

The DSPE-PEG₂₀₀₀-DTPA was synthetized as previously described [15]. Briefly, a solution of DSPE-PEG₂₀₀₀-NH₂ in DMSO (40.0 mg/ml) was added to DTPA dianhydride in DMSO:pyridine 7:3 (v/v) (32.0 mg/ml). The mixture was heated in an oil bath under constant stirring for 90 min at 100 °C. Then, ultrapure water was added to the reaction and mixture was maintained at 100 °C, for 90 min. The solvent was evaporated and the product was re-suspended in water and purified by dialysis using a Spectrapore^® membrane with a 1.0 kDa cut-off, at room temperature for 36 h. The final product was lyophilized in a 24 h cycle and stored at −20 °C.

2.3. Liposomes preparation

Liposomes composed of DOPE, CHEMS e DSPE-PEG₂₀₀₀ and DSPE-PEG₂₀₀₀-DTPA (SpHL-DTPA-PTX) at a molar ratio of 5.7:3.8:0.45:0.05, respectively, were prepared using the standard lipid film hydration method [3]. In brief, pre-determined chloroform aliquots of the lipids and PTX (0.5 mg/ml) were transferred to round bottom flask and a lipid film was obtained by evaporating the organic solvent under reduced pressure. Next, to promote the complete ionization of CHEMS molecules, an aliquot of NaOH solution (0.456 M) was added at a 1:1 molar ratio CHEMS:NaOH. The film was hydrated with NaCl 0.9% (w/v), followed by vigorous shaking in vortex. The vesicles were sonicated (20% amplitude) in an ice bath for 5 min using a high-intensity ultrasonic processor (R2D091109 model; Unique^® Instruments, Indaiatuba, Brazil). The suspension were submitted to a centrifugation process (Sigma 4k-15 centrifuge, Sigma Laborzentrifugen GmbH, Osterode, Germany) at 3000 rpm at 4 °C for 10 min to eliminate non-entrapped PTX. Since the drug shown low water solubility, it precipitate and a drug pellet is formed. The supernatant suspension represent the final and purified formulation. For the folate-coated liposomes, 0.05% of DSPE-PEG₂₀₀₀-folate was added to the lipid film formation.

2.4. Physicochemical characterization

2.4.1. Mean diameter and zeta potential

The mean diameter of SpHL-DTPA-PTX and SpHL-DTPA-folate-PTX was determined by dynamic light scattering (DLS) at 25 °C and at a fixed angle of 173°, using Zetasizer NanoZS90 (Malvern Instruments, England). The Zeta potential of the samples was measured by DLS associated to the electrophoretic mobility using the same equipment. All the samples were analyzed after 10-fold dilution in filtered NaCl 0.9% (w/v) solution (cellulose ester membrane, 0.45 mM, Millipore). Data were expressed as the mean ± standard deviation (SD) of at least three different batches.

2.4.2. Drug encapsulation percentage

The percentage of drug encapsulation of PTX in SpHL-DTPA-PTX or SpHL-DTPA-folate-PTX was determined by high performance liquid chromatography (HPLC) based on the determination of PTX concentration in the liposomes before (non-purified liposomes) and after centrifugation (purified liposomes) [24]. The liposomal vesicles were disrupted using isopropanol in a volume ratio of 1:10 and later diluted in a mixture of acetonitrile:water (55:45 v/v). This dispersion was filtered through a 0.45 μm Millex HV filter (Millipore, Billerica, MA, USA) and injected in the chromatographic apparatus. The encapsulation percentage (EP) was calculated using the following equation and the data were expressed as the mean ± standard deviation of at least three different liposomal formulation measurements:

$$EP=\frac{[PTX\text{] }purifiedliposomes}{\text{[}PTX\text{] }nonpurifiedliposomes}\times 100$$

2.5. Radiolabeling procedure

A 0.250 ml aliquot of the SpHL-DTPA-PTX or SpHL-folate-DTPAPTX was added to 200 μg of SnCl₂·2H₂O in acid solution (2 mg/ml) and the pH was adjusted for 7.4 in a sealed vial. An aliquot of 0.1 ml of Na^99mTcO₄ (37 MBq) was then added and the mixture was kept at room temperature for 15 min ^99mTc-PTX was prepared as reported by Monteiro et al. and it was used in this study as a comparative group with the liposomal preparations presented in this paper [26].

2.6. Radiochemical purity evaluation

Radiochemical purity analyses were performed by thin layer chromatography on silica gel (TLC-SG; Merck, Darmstadt, Germany) using acetone as mobile phase to quantify free ^99mTcO₄⁻. The radioactivity was determined by a gamma counter (Wallac Wizard 1470–020 Gamma Counter; PerkinElmer Inc., Waltham, Massachusetts, USA). The solution was purified from ^99mTcO₂ using a 0.22 μm syringe filter, as previously described [23,24].

2.7. In vitro stability: saline and plasma

TLC-SG was used to estimate the stability of ^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-folate-DTPA-PTX in the presence of NaCl 0.9% (w/v), at room temperature, or plasma, at 37 °C. For plasma stability, 90 μl of the ^99mTc-PTX solution were incubated with 1.0 ml of fresh mice plasma, under agitation, at 37 °C. Radiochemical stabilities were determined on samples taken up at 1, 2, 4, 6, 8, and 24 h after incubation.

2.8. Pharmacokinetic blood clearance

An aliquot of 100 μl of ^99mTc-SpHL-DTPA-PTX or ^99mTc-SpHL-fo-late-DTPA-PTX was administrated to healthy BALB/c female mice (n = 7) through the tail vein. A small incision was made in the distal tail and blood samples (~20 μl each) were collected at 5, 10, 15, 30, 45, 60, 90, 120, 240, 360, 480 and 1440 min after administration. Each sample was weighted, and the associated radioactivity was determined in an automatic scintillation apparatus. The percentage of injected activity per gram (%ID/g) in each sample was determined. The data were plotted as a function of time, expressed as mean ± SD.

2.9. Biodistribution

Biodistribution studies were carried out in healthy female BALB/c mice (n = 7). Aliquots of 3.7 MBq of ^99mTc-PTX, ^99mTc-SpHL-DTPAPTX or ^99mTc-SpHL-folate-DTPA-PTX were injected intravenously in the animals. At 4 and 8 h post-injection, mice were anesthetized with a mixture of xylazine (10 mg/kg) and ketamine (80 mg/kg). Liver, spleen, kidneys, stomach, heart, lungs, muscle, thyroid, and intestine were removed, and placed in pre-weighted plastic test tubes. The radioactivity was measured using an automatic gamma counter. A standard dose containing the same injected amount was counted simultaneously in a separate tube, which was defined as 100% radioactivity. The results were expressed as the percentage of injected dose per gram of tissue (%ID/g).

2.10. In vivo tumor model

MDA-MB-231 cells were cultivated in DMEM supplemented with 10% (v/v) fetal bovine serum, penicillin (100 IU/ml), and streptomycin (100.0 μg/ml) and kept at 37 °C in humidified air containing 5% CO₂. Cells were grown to confluence, then, harvested by trypsinization, centrifuged (5 min at 330 x g) and resuspended in 1.0 ml of DMEM. An aliquot of the cell suspension was stained with trypan blue (1:1) and counted in Newbauer chamber to determine cell viability.

An aliquot of 75 μl containing 5 × 10⁶ cells in DMEM were suspended in 75 μl of Matrigel^® and injected subcutaneously into the right thigh of female BALB/c nude mice (6–8 weeks) to established the human breast tumor xenograph [27]. Tumor cells were allowed to grow in vivo for 30 days and the animal was used for scintigraphic images.

2.11. Scintigraphic images

An aliquot of 18 MBq of ^99mTc-PTX, ^99mTc-SpHL-DTPA-PTX or ^99mTc-SpHL-folate-DTPA-PTX was injected intravenously to each healthy female BALB/c nude mouse or MDA-MB-231 tumor-bearing female BALB/c nude mouse (n = 7). Mice were anesthetized and horizontally placed under the collimator of a gamma camera (Mediso, Budapest, Hungary) coupled with a low-energy high-resolution collimator. Images were acquired at 4 and 8 h after injection using a 256 × 256 × 16 matrix size, with a 20% energy window set at 140 keV for a period of 300 s each. The same procedure was performed to tumor-bearing mice and images were acquired at 4 and 8 h post-administration (n = 4).

2.12. Statistical analysis

Data were expressed as the mean ± standard deviation (SD). Normality and homogeneity of variance analysis were performed by D’agostino and Pearson and Bartlett’s tests. Physical-chemical data were tested using the test t de student, while biodistribution data and the one-way analysis of variance (ANOVA) test followed by Tukey’s test. A P-value less than 0.05 were considered to indicate a significant difference. All data were analyzed by GraphPad PRISM, version 5.00 software (GraphPad Software Inc., La Jolla, California, USA).

3. Results

3.1. Physicochemical characterization

SpHL-DTPA-PTX and SpHL-folate-DTPA-PTX showed a mono-disperse size distribution, a neutral zeta potential and a similar encapsulation percentage (Table 1). These data support that both delivery systems presented suitable characteristics for intravenous administration.

Table 1.

Physicochemical characterization of liposomal systems (n = 3).

Formulation	Diameter (nm)	PI	Zeta Potential (mV)	Encapsulation Percentage (%)	Drug load (mg/mL)
SpHL-DTPA-PTX	145 ± 3	0.232 ± 0.02	−0.34 ± 2.7	84 ± 1.5	0.42 ± 0.01
SpHL-folate-DTPA-PTX	152 ± 7	0.252 ± 0.01	−1.69 ± 0.8	86 ± 2.2	0.43 ± 0.01

The results were expressed as the mean ± standard deviation.

3.2. Radiochemical purity and in vitro stability

The radiolabeled yields for ^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-folate-DTPA-PTX were 98.4 ± 1.1%and 98.0 ± 1.0%, respectively. ^99mTcO₂ produced in the reaction was removed by passing the system through sterile filters (pore diameter = 0.22 μm) since this radiocolloid remains in the filter while ^99mTcO₄⁻ and the radiolabeled complexes are freely filtered [23,24].^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-fo-late-DTPA-PTX showed high stability in saline and plasma, even after long periods (Fig. 1). At 24 h post-incubation, in both media, the radiochemical purity was higher than 90% for both radiolabeled complexes, which indicates labeling stability between the ^99mTc and DTPA.

Fig. 1.

In vitro stability of ^99mTc-SpHL-DTPA-PTX (A) and ^99mTc-SpHL-folate-DTPA-PTX (B) as a function of time in presence of saline at room temperature (square) and plasma at 37 °C (circle) (n = 5).

3.3. Pharmacokinetic blood clearance

After intravenous administration in healthy female BALB/c mice, blood levels of ^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-folate-DTPAPTX declined in a monophasic manner

showing half-life of 400.1 and 541.8 min, respectively. The areas under the curve (AUC) were also calculated for ^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-folate-DTPA-PTX (1245.04% ID min⁻¹ and 1637.68% ID min⁻¹, respectively).

3.4. Scintigraphic images and biodistribution studies

Scintigraphic images were acquired at 4 and 8 h after ^99mTc-PTX, ^99mTc-SpHL-DTPA-PTX or ^99mTc-SpHL-folate-DTPA-PTX administration in healthy female BALB/c mice (Fig. 2). Liver and spleen represent the major site of accumulation for all the evaluated complexes. Renal excretion was also observed for all systems. In contrast to the liposomes, ^99mTc-PTX showed a significant intestinal accumulation.

Fig. 2.

Scintigraphic images obtained at 4 and 8 h post-injection of ^99mTc-PTX (A),^99mTc-SpHL-DTPA-PTX (B) and ^99mTc-SpHL-folate-DTPA-PTX (C) in healthy female BALB/c mice.

Biodistribution studies corroborate the findings in scintigraphic images (Fig. 3). ^99mTc-PTX, ^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-folate-DTPA-PTX showed high uptake by the liver, spleen and kidneys and no significant accumulation was verified in other organs, such as stomach and thyroid, which indicates radiolabeled stability. As afore mentioned, ^99mTc-PTX (Fig. 3A) showed significant intestine uptake.

Fig. 3.

Biodistribution profile obtained at 4 h (white bars) and 8 h (black bars) post-injection of ^99mTc-PTX (A), ^99mTc-SpHL-DTPA-PTX (B) and ^99mTc-SpHL-folate-DTPA-PTX(C) in healthy female BALB/c mice. Bars represent the mean percentage of the injected dose per gram of tissue ± SD (n = 7).

In order to reduce the number of animals used in this study, tumor-bearing mice were used only for imaging acquisition. Scintigraphic images provide information, in real time, of the whole body distribution of radiolabeled nanoparticles preventing the unnecessary use of a large amount of animals. Images were acquired at 4 and 8 h post-injection of ^99mTc-PTX, ^99mTc-SpHL-DTPA-PTX or ^99mTc-SpHL-folate-DTPA-PTX in MDA-MB-231 tumor-bearing nude mice. The results showed the same biodistribution profile than those for healthy mice, indicating that the presence of the tumor tissue did not alter the distribution of the formulations. In summary, high uptake was observed in liver, spleen, and kidneys (Fig. 4). In addition, the tumor could be clearly seen after administrating ^99mTc-SpHL-DTPA-PTX or ^99mTc-SpHL-folate-DTPA-PTX. However, by injecting ^99mTc-PTX, the tumor area was marginally visualized in the images. The tumor-to-muscle ratios confirm the higher accumulation for liposomal formulations than ^99mTc-PTX (Fig. 5). At 4 h post-injection, 99mTc-SpHL-folate-DTPA-PTX showed significant higher tumor-to-muscle ratios than the other complexes.

Fig. 4.

Scintigraphic images obtained at 4 and 8 h post-injection of ^99mTc-PTX(A), ^99mTc-SpHL-DTPA-PTX(B) and ^99mTc-SpHL-folate-DTPA-PTX(C) in xenographic tumor-bearing female BALB/c mice.

Fig. 5.

Tumor-to-muscle ratios determined by scintigraphic images at 4 and 8 h post-injection of ^99mTc-PTX (white bars), ^99mTc-SpHL-DTPA-PTX (gray bars) and ^99mTc-SpHL-folate-DTPA-PTX (black bars) in xenographic tumor-bearing mice. Asterisks indicate significant difference (*p < 0.05; ***p < 0.001).

4. Discussion

Paclitaxel (PTX) is one of the most effective and potent antitumor drugs used in clinical oncology against a wide range of solid tumors, including metastatic breast cancer [6,8,10,26,28]. However, its low specificity can lead to an increased systemic toxicity, since the drug can act indiscriminately in tumor and healthy tissues. Serious hypersensitivity reactions, peripheral sensory neuropathy, and myelosuppression are the most commons side effects, which can limit its clinical application. Due to its poor water solubility, PTX is dispersed in a mixture of dehydrated ethanol and Cremophor EL^® (1:1 v/v), that is also a toxic vehicle, and contributes to PTX toxicity [2,3,5,6,9–11].

Many efforts have been made to develop new strategies for cancer diagnosis and treatment that minimize dose-limiting toxicity [1,4,29,30]. Liposomes have emerged as an interesting alternative for drug delivery since this system can encapsulate both lipophilic and hydrophilic drugs. Additionally, liposomes can protect encapsulated drugs and improve treatment efficacy [6,12,13,28,29]. In this study, SpHL-folate-PTX was used to selectively deliver PTX into tumor, since in vitro assays indicated that folate-coated strategy has dramatically improved the cytotoxicity of pH-sensitivity liposomes in comparison to either non-functionalized liposomes or micellar dispersion of PTX [3]. These encouraging results lead to further in vivo analysis seeking to understand the biodistribution profile, which is essential to evaluate the real contribution of the new targeted-formulation in achieving the tumor.

SpHL-PTX and SpHL-foalte-PTX have interesting characteristics that confers to the system specific abilities to accumulate in the tumor region, such as long-circulation, pH-sensibility and specific targeting to cell receptors. According to the American Pharmacopoeia [31], nanocarriers to be injected intravenously must be mean diameter equal or less than 500 nm, in order to guarantee the stability of an injectable lipid formulation. In addition, to prevent pulmonary embolism and microvasculature occlusion, the percentage concentration (w/v) of vesicles greater than 5 μm should be less than 0.05% of the total dispersed phase [32,33]. Besides that, the reduced particle size (≤500 nm) also favors passive targeting of liposomes allowing the extravasation and retention of nanostructures into tumors by EPR effect [34–37].

Our results by DLS showed that there is no difference between the size and PDI of both liposomal systems prepared. Reduced size vesicles and homogeneous distribution of liposomal systems prevent the occurrence of aggregation and fusion of the vesicles. In addition,particles ranging from 50 to 200 nm might be less uptaken by SMF cells after the systemic administration Ulrich [38]; Yang et al. [39]. DLS associated to the electrophoretic mobility was used to determine surface charge of liposomes, SpHL-folate-PTX and SpHL-PTXshowed a zeta potential near to neutral. Similar results were previous obtained, and could be explained by the presence of PEG that generates a hydrophilic layer that impart liposome mobility. The presence of PEG is pivotal to protect the vesicles from fusion/aggregation and prevent opsonization [3,40–42]. Therefore, SpHL-folate-PTX and SpHL-PTX showed physicochemical characteristics that allow further in vivo studies.

^99mTc-PTX was prepared using the methodology described by Monteiro et al. [14,26] and was used as a comparative group with the liposomal preparations presented in these studies [3,16]. For liposome radiolabeling, the lipid DSPE-PEG₂₀₀₀-DTPA was used since it is well known that ^99mTc generates a stable complex with chelating agents, such as DTPA, for image purposes [23,41,43,44]. The results showed a high radiochemical purity and stability (over to 90%), up to 24 h after radiolabeling. Radiochemical purity above 90% is required since the presence of high amount of impurities leads to poor image quality, which might result in a biodistribution profile that no longer reflects the nanoparticle behavior [26,45].

^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-folate-DTPA-PTX showed long blood circulation times that is important to confer more passages through the tumor area. Tumor vessels have been shown to have larger pores than the regular vasculature; consequently, particles in nano-metric size, such as liposomes, are able pass through the walls of a vessel into the surrounding tissues, e.g. tumor [23,26,46].

Nanoparticles are extensively phagocytosed by macrophages present in the liver and spleen organs [25,26,46,47]. As predicted, the biodistribution studies showed high uptake in liver and spleen for ^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-folate-DTPA-PTX. The kidneys also showed a significant uptake, which indicates renal clearance of all complexes. Noteworthy, the low uptake in thyroid and stomach for ^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-folate-DTPA-PTX confirms the high labeling stability showed by in vitro assays [46–49]. In addition, as previously described, ^99mTc-PTX showed a substantial uptake by the intestines, suggesting that fecal route also contributes to the excretion of ^99mTc-PTX [26].

The formulations evaluated in these studies showed a significant tumor uptake compared with the contralateral muscle in all timeframes. ^99mTc-SpHL-DTPA-PTX and 99mTc-SpHL-folate-DTPA-PTX showed higher tumor-to-muscle ratio than ^99mTc-PTX, at 8 h post-injection, indicating that longer circulation achieved by the liposomes leads to higher tumor accumulation. Interestingly, 99mTc-SpHL-folate-DTPAPTX showed significant higher tumor-to-muscle ratio than ^99mTc-SpHLDTPA-PTX and ^99mTc-PTX at 4 h after administration, suggesting that the folate-coating strategy can favor tumor accumulation in shorter time. The presence of folate on the liposome surface might contribute to the tumor uptake since MDA-MB-231 cells overexpress folate receptor on their membrane [49,50]. Therefore, ^99mTc-SpHL-folate-DTPA-PTX showed better performance than ^99mTc-SpHL-DTPA-PTX and ^99mTc-PTX, since short time accumulation is interesting to both approaches, diagnosis and therapy. Imaging probes that quickly achieve higher signal-to-noise ratios are preferable and can ultimately lead to more optimal patient care. Additionally, nanoparticles that can deliver the drug through longer periods are adequate to guarantee sustained drug concentration in the tumor.

Therefore, our hypothesis is that 99mTc-SpHL-folate-DTPA-PTX might enhance the drug concentration into the cytoplasmic of tumor cells, since the liposomes intoendosomal vesicles (pH 5.0) will undergo destabilization or fusion with endosomal bilayer, releasing PTX directly in cytoplasm cell [3,14,37,51]. Moreover, folate will provide a cell-specific intracellular delivery, once cancer cells are known to over-express a number of cell surface receptors, such as folate receptors [3,49,50]. Indeed, higher tumor accumulation was achieved by using folate-coated liposomes that might be interesting to improve antitumor efficacy.

In conclusion, liposomes showed adequate mean diameter and polidispersivity that allows intravenous administration. Moreover, ^99mTc-SpHL-DTPA-PTX and ^99mTc-SpHL-folate-DTPA-PTX showed high tumor uptake in scintigraphic images. Special emphasis should be given to ^99mTc-SpHL-folate-DTPA-PTX that showed a sustained and higher tumor-to-muscle ratio than non-functionalized liposome. Therefore, results presented in this study indicate the feasibility of ^99mTc-SpHL-folate-DTPA-PTX as a PTX delivery system to folate positive tumors.