“Smart” Drug Carriers: PEGylated TATp-Modified pH-Sensitive Liposomes

Introduction

Intracellular delivery of therapeutic actives or biologics is a challenging task in drug delivery. Nanocarriers like liposomes, micelles, and nano-particles/emulsions have been frequently used for delivery or targeting (Moghimi et al., 2005; Torchilin, 2006). These carriers have the ability to accumulate in specific organ via the EPR effect. Ideally, one would like to see these nanocarriers and their cargos to be then delivered inside individual cells in the target area.

Earlier, we have described a principal construction of multifunctional “smart” drug carrier systems comprising a cell-penetrating function, TATp, and a protective pH-sensitive polymeric coating formed by PEG-HZ-PE (Sawant et al., 2006). The protective pH-sensitive PEG coating has the ability to sterically shield the TATp function and prevent it from the non-specific internalization while in circulation. The TATp can become exposed however at the desired sites with lowered pH values, such as tumors or infarcts, due to the removal of PEG coat, which should result in the enhanced internalization of the nanocarriers into target cells.

Here, we present our data on the sequential characterization of multifunctional pH-sensitive liposomal nanocarriers using in vitro (pH-dependant degradation), in situ (hypoxic acidosis), and in vivo (tumor acidosis) tests.

Materials and Methods

Methods

Synthesis of mPEG₂₀₀₀-HZ-PE and TATp-PEG₁₀₀₀-PE conjugates

mPEG₂₀₀₀-HZ-PE was synthesized in three steps. First, mPEG₂₀₀₀-SH was reacted with SMCCHz to form an acyl hydrazide-activated PEG, while aromatic ketone-derivatized phospholipid was synthesized by reacting 4-acetyl phenyl maleimide with phosphothioethanolamine. Then, acyl hydrazide-activated PEG and aromatic ketone-derivatized lipid were reacted to form mPEG₂₀₀₀-HZ-PE (see the Scheme 1). TATp-PEG₁₀₀₀-PE was synthesized using a heterobifunctional NHS-PEG₁₀₀₀-Mal derivative, DOPE-NH₂ and TATp-cysteine (Kale and Torchilin, 2007a, b).

Scheme 1

Synthesis of mPEG-HZ-PE conjugate.

Preparation of the Liposomal Formulations

The pH-sensitive or pH-insensitive, Rh-labeled, TATp-bearing liposomes were prepared by the lipid film hydration method. A mixture of PC:Chol (7:3), TATp-PEG₁₀₀₀-PE, Rh-PE and either mPEG₂₀₀₀-HZ-PE (pH-sensitive) or mPEG₂₀₀₀-DSPE (pH-insensitive) at molar ratio 10:0.25:0.1:15 was evaporated under reduced pressure. The dry lipid formed was hydrated with phosphate buffer saline, pH 7.4. The liposomal suspension was filtered through 0.2 μm polycarbonate filters and stored at 4°C until use. The liposome particle mean size and size distribution were observed using a Coulter N4 Plus submicron particle analyzer.

In vitro pH-dependent Degradation of mPEG₂₀₀₀-HZ-PE conjugates

The time-dependent degradation of mPEG-HZ-PE micelles incubated in buffer solutions (phosphate buffer saline, pH 7.4 and pH 5.5) maintained at 37°C was followed by HPLC using Shodex KW-804 size exclusion column (Kale and Torchilin, 2007a, b). The degradation kinetics of micelles was assessed by following the area under the micelle curve.

Avidin-biotin Affinity Chromatography

To check their pH-sensitivity, biotin-containing micelles were formulated by mixing mPEG₂₀₀₀-HZ-PE (60 mol%), PEG₇₅₀-PE (37 mol%), Rhodamine-PE (0.5 mol%, fluorescent marker), and biotin-PE (2.5 mol%, biotin component) in chloroform. Chloroform was evaporated and a thin film was formed using rotary evaporator. To test the binding of biotin-bearing Rh-PE-labeled, TATp-modified liposomes before and after the incubation at lowered pH values, the corresponding samples were kept for 3 h at pH 7.4 or pH 5.5 and then applied onto the Immobilized NeutrAvidin protein column. The degree of the retention of the corresponding preparation on the column was estimated following the decrease in the sample rhodamine fluorescence at 550/590 nm after passing through the NeutrAvidin column (Sawant et al., 2006).

In vitro Cell Culture Study

H9C2 rat embryonic cardiomyocytes were grown on coverslips in 6-well plates in 10% fetal bovine serum DMEM, then treated with various Rh-PE-labeled liposome samples (with and without preincubation for 3 h at pH 5.5) in serum-free medium (2 ml/well, 30 mg total lipid/ml). After a 1 h incubation period, the medium was removed, and the plates washed with serum-free medium three times. Individual cover-slips were mounted cell-side down onto fresh glass slides with PBS. Cells were viewed with a Nikon Eclipse E400 microscope under bright light or under epifluorescence with rhodamine/TRITC filter (Sawant et al., 2006). The images were analyzed using ImageJ 1.34I software (NIH) for integrated density comparison of red fluorescence between two groups.

In situ Study

An isolated, isovolumic (balloon-in-left ventricle (LV)) rat heart preparation was set-up using Langendorff assembly as described previously (Verma et al., 2005). The Rh-labeled, TATp-bearing liposomal formulations (pH-sensitive or pH-insensitive) were infused over a 1 min period, prior to the onset of ischemia. Hypoxic and normoxic tissue sections were distinguished using nitroblue tetrazolium (NBT) staining. The hypoxic tissue sections were cryo-fixed after hardening overnight at −80°C in Tissue-Tek (Ted Palla Inc, Redding, CA). The sections were cut on Microtome Plus TBS (Triangular Biomedical Sciences Inc, Durham, NC). The sections were washed several times in phosphate buffer saline (pH 7.4), dried and fixed on slides using Fluor Mounting media (Trevigen, Gaithersberg, MD). The fixed sections were observed under fluorescence microscope with TRITC filter.

In vivo Study

LLC tumors were grown in nu/nu mice (Charles River Breeding Laboratories, MA) by the subcutaneous.injection of 8 × 10 ^{LLC cells per mouse into the left flank (protocol # 05–1233R, approved by the Institutional Animal Care and Use Committee at Northeastern University, Boston). When tumor reached 5–10 mm in diameter, they were injected at four to five different spots with 150 μl of Rh-labeled, TATp-bearing pH-sensitive or pH-insensitive liposomes in phosphate buffered saline, pH 7.4. Mice were killed 6 h later by cervical dislocation, and excised tumors were cryo-fixed as described above. Microtome cut sections were washed thoroughly with PBS (pH 7.4), dried and fixed on slides using Fluor Mounting medium. These sections were observed under fluorescence microscopy using TRITC filter (}Torchilin et al., 2003). Further, the images were analyzed using ImageJ 1.34I software (NIH) for integrated density comparison of red fluorescence between pH-sensitive and pH-insensitive groups.

Materials

Egg phosphatidylcholine (egg PC), cholesterol (Ch), mPEG₂₀₀₀-DSPE, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), Rhodamine-PE (Rh-PE), and phosphatidylthioethanolamine (DPPE-SH) were purchased from Avanti Polar Lipids (Alabaster, AL); mPEG₂₀₀₀-SH was from Nektar Therapeutics (Huntsville, AL), maleimide-PEG₁₀₀₀-NHS from Quanta Biode-sign (Powell, OH), and TATp-cysteine from Research Genetics (Huntsville, AL). Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate hydrazide (SMCCHz) was purchased from Molecular Biosciences (Boulder, CO). 4-acetyl phenyl maleimide, Sephadex G25m, and Sepharose CL4B were purchased from Sigma-Aldrich. Lewis Lung Carcinoma (LLC) cell line was from ATCC (Rockville, MD). Delbecco's minimal essential medium, complete serum free medium and fetal bovine serum were purchased from Cellgro (Kansas City, MO).

Methods

Synthesis of mPEG₂₀₀₀-HZ-PE and TATp-PEG₁₀₀₀-PE conjugates

mPEG₂₀₀₀-HZ-PE was synthesized in three steps. First, mPEG₂₀₀₀-SH was reacted with SMCCHz to form an acyl hydrazide-activated PEG, while aromatic ketone-derivatized phospholipid was synthesized by reacting 4-acetyl phenyl maleimide with phosphothioethanolamine. Then, acyl hydrazide-activated PEG and aromatic ketone-derivatized lipid were reacted to form mPEG₂₀₀₀-HZ-PE (see the Scheme 1). TATp-PEG₁₀₀₀-PE was synthesized using a heterobifunctional NHS-PEG₁₀₀₀-Mal derivative, DOPE-NH₂ and TATp-cysteine (Kale and Torchilin, 2007a, b).

Scheme 1

Synthesis of mPEG-HZ-PE conjugate.

Synthesis of mPEG₂₀₀₀-HZ-PE and TATp-PEG₁₀₀₀-PE conjugates

mPEG₂₀₀₀-HZ-PE was synthesized in three steps. First, mPEG₂₀₀₀-SH was reacted with SMCCHz to form an acyl hydrazide-activated PEG, while aromatic ketone-derivatized phospholipid was synthesized by reacting 4-acetyl phenyl maleimide with phosphothioethanolamine. Then, acyl hydrazide-activated PEG and aromatic ketone-derivatized lipid were reacted to form mPEG₂₀₀₀-HZ-PE (see the Scheme 1). TATp-PEG₁₀₀₀-PE was synthesized using a heterobifunctional NHS-PEG₁₀₀₀-Mal derivative, DOPE-NH₂ and TATp-cysteine (Kale and Torchilin, 2007a, b).

Scheme 1

Synthesis of mPEG-HZ-PE conjugate.

Preparation of the Liposomal Formulations

The pH-sensitive or pH-insensitive, Rh-labeled, TATp-bearing liposomes were prepared by the lipid film hydration method. A mixture of PC:Chol (7:3), TATp-PEG₁₀₀₀-PE, Rh-PE and either mPEG₂₀₀₀-HZ-PE (pH-sensitive) or mPEG₂₀₀₀-DSPE (pH-insensitive) at molar ratio 10:0.25:0.1:15 was evaporated under reduced pressure. The dry lipid formed was hydrated with phosphate buffer saline, pH 7.4. The liposomal suspension was filtered through 0.2 μm polycarbonate filters and stored at 4°C until use. The liposome particle mean size and size distribution were observed using a Coulter N4 Plus submicron particle analyzer.

In vitro pH-dependent Degradation of mPEG₂₀₀₀-HZ-PE conjugates

The time-dependent degradation of mPEG-HZ-PE micelles incubated in buffer solutions (phosphate buffer saline, pH 7.4 and pH 5.5) maintained at 37°C was followed by HPLC using Shodex KW-804 size exclusion column (Kale and Torchilin, 2007a, b). The degradation kinetics of micelles was assessed by following the area under the micelle curve.

Avidin-biotin Affinity Chromatography

To check their pH-sensitivity, biotin-containing micelles were formulated by mixing mPEG₂₀₀₀-HZ-PE (60 mol%), PEG₇₅₀-PE (37 mol%), Rhodamine-PE (0.5 mol%, fluorescent marker), and biotin-PE (2.5 mol%, biotin component) in chloroform. Chloroform was evaporated and a thin film was formed using rotary evaporator. To test the binding of biotin-bearing Rh-PE-labeled, TATp-modified liposomes before and after the incubation at lowered pH values, the corresponding samples were kept for 3 h at pH 7.4 or pH 5.5 and then applied onto the Immobilized NeutrAvidin protein column. The degree of the retention of the corresponding preparation on the column was estimated following the decrease in the sample rhodamine fluorescence at 550/590 nm after passing through the NeutrAvidin column (Sawant et al., 2006).

In vitro Cell Culture Study

H9C2 rat embryonic cardiomyocytes were grown on coverslips in 6-well plates in 10% fetal bovine serum DMEM, then treated with various Rh-PE-labeled liposome samples (with and without preincubation for 3 h at pH 5.5) in serum-free medium (2 ml/well, 30 mg total lipid/ml). After a 1 h incubation period, the medium was removed, and the plates washed with serum-free medium three times. Individual cover-slips were mounted cell-side down onto fresh glass slides with PBS. Cells were viewed with a Nikon Eclipse E400 microscope under bright light or under epifluorescence with rhodamine/TRITC filter (Sawant et al., 2006). The images were analyzed using ImageJ 1.34I software (NIH) for integrated density comparison of red fluorescence between two groups.

In situ Study

An isolated, isovolumic (balloon-in-left ventricle (LV)) rat heart preparation was set-up using Langendorff assembly as described previously (Verma et al., 2005). The Rh-labeled, TATp-bearing liposomal formulations (pH-sensitive or pH-insensitive) were infused over a 1 min period, prior to the onset of ischemia. Hypoxic and normoxic tissue sections were distinguished using nitroblue tetrazolium (NBT) staining. The hypoxic tissue sections were cryo-fixed after hardening overnight at −80°C in Tissue-Tek (Ted Palla Inc, Redding, CA). The sections were cut on Microtome Plus TBS (Triangular Biomedical Sciences Inc, Durham, NC). The sections were washed several times in phosphate buffer saline (pH 7.4), dried and fixed on slides using Fluor Mounting media (Trevigen, Gaithersberg, MD). The fixed sections were observed under fluorescence microscope with TRITC filter.

In vivo Study

LLC tumors were grown in nu/nu mice (Charles River Breeding Laboratories, MA) by the subcutaneous.injection of 8 × 10 ^{LLC cells per mouse into the left flank (protocol # 05–1233R, approved by the Institutional Animal Care and Use Committee at Northeastern University, Boston). When tumor reached 5–10 mm in diameter, they were injected at four to five different spots with 150 μl of Rh-labeled, TATp-bearing pH-sensitive or pH-insensitive liposomes in phosphate buffered saline, pH 7.4. Mice were killed 6 h later by cervical dislocation, and excised tumors were cryo-fixed as described above. Microtome cut sections were washed thoroughly with PBS (pH 7.4), dried and fixed on slides using Fluor Mounting medium. These sections were observed under fluorescence microscopy using TRITC filter (}Torchilin et al., 2003). Further, the images were analyzed using ImageJ 1.34I software (NIH) for integrated density comparison of red fluorescence between pH-sensitive and pH-insensitive groups.

Results and Discussion

Avidin-biotin Affinity Chromatography

To determine the pH-sensitivity of mPEG-HZ-PE conjugates, model biotin-containing micelles with the biotin moieties shielded by the pH-cleavable mPEG₂₀₀₀-HZ-PE were eluted through the column with the immobilized biotin. The pH-cleavable micelle formulation incubated at pH 7.4 at 37°C for 3 h showed only a minimal biotin-mediated binding against ca. 70% binding of the same micelle formulation incubated at pH 5.5 at 37°C for 3 h, Fig. 1. This clearly shows the shielding effect of mPEG₂₀₀₀-HZ-PE conjugate under the physiological pH condition and the removal of PEG chains and bioting deshielding after the exposure to acidic environment.

Figure 1

Binding of pH-sensitive biotin-micelles to NeutrAvidin columns after incubation at room temperature at pH 5.5 and 7.4 for 3 h.

In Vitro Cell Culture Study

To study shielding/deshielding effect of mPEG-HZ-PE under the influence of acidic pH, internalization of Rh-labeled, TATp-bearing, mPEG-HZ-PE shielded liposomes preincubated at pH 7.4 and pH 5.5 was followed using H9C2 cells. As seen in Fig. 2a and 2b, Rh-labeled, TATp-bearing, pH-sensitive liposomes incubated at pH 5.5 showed 2.5 times (ImageJ 1.34I data) more red fluorescence inside the cells than when incubated at pH 7.4 because of better accessibility of TATp for its action after the detachment of pH-sensitive PEG corona from liposomal surface under the influence of lowered pH.

Figure 2

Fluorescence microscopy showing internalization of Rh-PE-labeled/TATp/pH-sensitive liposomes by H9C2 cells after the liposome pre-incubation at pH 7.4 (a) and pH 5.5 (b).

In Situ Study

The lowered pH of hypoxic cardiac tissues undergoing the ischemia/infarction was used to test the pH-sensitivity of our nanocarriers. The liposomal formulations were infused for 1 min before inducing ischemia following normoxia-ischemia-reperfusion protocol in Langendorff's isolated rat heart model established in our laboratory. Rh-labeled, TATp-bearing, pH-sensitive liposomes showed significantly better accumulation of rhodamine fluorescence (i.e., rhodamine-labeled liposomes) inside myocardiocytes in the hypoxic area (when observed under fluorescence microscopy using TRITC filter), while their counterpart non-cleavable liposomes showed only a minimal accumulation of rhodamine fluorescence inside the cells in the hypoxic region (Figs. 3a and 3b). Non-cleavable liposomes accumulate in the interstitium and are not taken well by the cells because of shilded TATp function. These liposomes are removed during the washing procedure. The lowered pH inside the hypoxic tissues caused the removal of the pH-sensitive PEG corona thus exposing the earlier hidden TATp function and allowing for the enhanced penetration of the liposomes inside the cells the hypoxic region of heart, and they are not removed from inside the cells during any washing procedures.

Figure 3

Merged images of hypoxic myocardial tissue sections after the infusion in Langendorff isolated rat heart of Rh-labeled/TATp/pH-insensitive liposome (a) or pH-sensitive liposomes (b).

In Vivo Study

Trying to cover different physiological conditions, we attempted intratumoral injections of Rh-labeled, TATp-bearing, pH-sensitive or pH-insensitive liposomes into LLC tumor bearing mice. The lowered (slightly acidic) pH at the tumor sites is a well known fact, which is of interest while developing physiology-based targeted delivery systems. Under the fluorescence microscope with TRITC filter, samples prepared 6 h post-injection from tumors injected with TATp-bearing, Rh-labeled, pH-sensitive liposomes demonstrated intensive and bright red fluorescence which was 4 times (as per ImageJ 1.34I data) higher than that observed in the samples obtained from the tumors injected with TATp-bearing, Rh-labeled, pH-insensitive liposomes (Figs. 4a and 4b). The mechanism behind those differences is similar to the one described in the previous paragraph (here, non-internalized by the cells non-pH-sensitive liposomes are also removed from tissue samples as a result of the extensive washing procedure)

Figure 4

TRITC image of frozen tumor tissue section treated after the intratumoral injection o Rh-labeled/TATp/pH-insensitive liposome (a) or Rh-labeled/TATp/pH-sensitive liposome (b) in LLC tumor-bearing mice.

Synthesis and In vitro pH-dependant Degradation mPEG₂₀₀₀-HZ-PE Conjugates

As descried earlier, aromatic ketone-derived hydrazone-based PEG-PE conjugates were synthesized and evaluated for their pH-dependant degradation (Kale and Torchilin, 2007b). The presence of a methyl group (electron donating) on carbonyl functional group provides sufficient lability of hydrazone bond under mildly acidic conditions while an immediate aromatic ring (electron withdrawing) next to hydrazone bond offers stability under acidic and neutral conditions. mPEG₂₀₀₀-HZ-PE conjugate exhibited half-life of more than 40 h at pH 7.4 and 3.0 h at pH 5.5.

Avidin-biotin Affinity Chromatography

To determine the pH-sensitivity of mPEG-HZ-PE conjugates, model biotin-containing micelles with the biotin moieties shielded by the pH-cleavable mPEG₂₀₀₀-HZ-PE were eluted through the column with the immobilized biotin. The pH-cleavable micelle formulation incubated at pH 7.4 at 37°C for 3 h showed only a minimal biotin-mediated binding against ca. 70% binding of the same micelle formulation incubated at pH 5.5 at 37°C for 3 h, Fig. 1. This clearly shows the shielding effect of mPEG₂₀₀₀-HZ-PE conjugate under the physiological pH condition and the removal of PEG chains and bioting deshielding after the exposure to acidic environment.

Figure 1

Binding of pH-sensitive biotin-micelles to NeutrAvidin columns after incubation at room temperature at pH 5.5 and 7.4 for 3 h.

In Vitro Cell Culture Study

To study shielding/deshielding effect of mPEG-HZ-PE under the influence of acidic pH, internalization of Rh-labeled, TATp-bearing, mPEG-HZ-PE shielded liposomes preincubated at pH 7.4 and pH 5.5 was followed using H9C2 cells. As seen in Fig. 2a and 2b, Rh-labeled, TATp-bearing, pH-sensitive liposomes incubated at pH 5.5 showed 2.5 times (ImageJ 1.34I data) more red fluorescence inside the cells than when incubated at pH 7.4 because of better accessibility of TATp for its action after the detachment of pH-sensitive PEG corona from liposomal surface under the influence of lowered pH.

Figure 2

Fluorescence microscopy showing internalization of Rh-PE-labeled/TATp/pH-sensitive liposomes by H9C2 cells after the liposome pre-incubation at pH 7.4 (a) and pH 5.5 (b).

In Situ Study

The lowered pH of hypoxic cardiac tissues undergoing the ischemia/infarction was used to test the pH-sensitivity of our nanocarriers. The liposomal formulations were infused for 1 min before inducing ischemia following normoxia-ischemia-reperfusion protocol in Langendorff's isolated rat heart model established in our laboratory. Rh-labeled, TATp-bearing, pH-sensitive liposomes showed significantly better accumulation of rhodamine fluorescence (i.e., rhodamine-labeled liposomes) inside myocardiocytes in the hypoxic area (when observed under fluorescence microscopy using TRITC filter), while their counterpart non-cleavable liposomes showed only a minimal accumulation of rhodamine fluorescence inside the cells in the hypoxic region (Figs. 3a and 3b). Non-cleavable liposomes accumulate in the interstitium and are not taken well by the cells because of shilded TATp function. These liposomes are removed during the washing procedure. The lowered pH inside the hypoxic tissues caused the removal of the pH-sensitive PEG corona thus exposing the earlier hidden TATp function and allowing for the enhanced penetration of the liposomes inside the cells the hypoxic region of heart, and they are not removed from inside the cells during any washing procedures.

Figure 3

Merged images of hypoxic myocardial tissue sections after the infusion in Langendorff isolated rat heart of Rh-labeled/TATp/pH-insensitive liposome (a) or pH-sensitive liposomes (b).

In Vivo Study

Trying to cover different physiological conditions, we attempted intratumoral injections of Rh-labeled, TATp-bearing, pH-sensitive or pH-insensitive liposomes into LLC tumor bearing mice. The lowered (slightly acidic) pH at the tumor sites is a well known fact, which is of interest while developing physiology-based targeted delivery systems. Under the fluorescence microscope with TRITC filter, samples prepared 6 h post-injection from tumors injected with TATp-bearing, Rh-labeled, pH-sensitive liposomes demonstrated intensive and bright red fluorescence which was 4 times (as per ImageJ 1.34I data) higher than that observed in the samples obtained from the tumors injected with TATp-bearing, Rh-labeled, pH-insensitive liposomes (Figs. 4a and 4b). The mechanism behind those differences is similar to the one described in the previous paragraph (here, non-internalized by the cells non-pH-sensitive liposomes are also removed from tissue samples as a result of the extensive washing procedure)

Figure 4

TRITC image of frozen tumor tissue section treated after the intratumoral injection o Rh-labeled/TATp/pH-insensitive liposome (a) or Rh-labeled/TATp/pH-sensitive liposome (b) in LLC tumor-bearing mice.

Conclusions

The development of the targeted drug carriers carrying the temporarily hidden function (e.g., cell penetrating peptide, TATp), and a detachable PEG-HZ-PE which, in addition to prolonging circulation half-life of carriers, can expose TATp function only under the action of certain local stimuli (such as lowered pH), represent a significant step on the way toward “smart” multifunctional pharmaceutical nanocarriers for target accumulation by EPR effect and intracellular penetration in a controlled fashion.

Acknowledgments