Multimeric peptide-based PEG nanocarriers with programmable elimination properties

Abstract

In the current study, the design, synthetic feasibility and biochemical characterization of biodegradable peptidic PEG-based nanocarriers are described. The components were selected to influence the body elimination pathway upon nanocarrier biodegradation. Two prototypical nanocarriers were prepared using non-PEGylated and PEGylated peptidic cores [CH(3)CO-(Lys-betaAla-betaAla)(X)-Cys-CONH(2) (X=2, 4)]. A homodimeric nanocarrier with 4 copies of fluorescein-PEG5kDa was synthesized by linking two PEGylated peptidic cores (X=2) using a disulfide bond. A dual labeled heterodimeric nanocarrier with 2 copies of fluorescein-PEG5kDa and 4 copies of Texas Red was also synthesized. Optimum conditions for linking imaging agents, PEG, or a peptidic core to a peptidic core were determined. Significantly higher yields (69% versus 30%) of the PEGylated peptidic core were obtained by using 2 copies of beta-alanine as a spacer along with increasing DMSO concentrations, which resulted in reduced steric hindrance. Stoichiometric addition of the components was also demonstrated and found to be important for reducing polydispersity. Nanocarrier biodegradation was evaluated in simulated intracellular and extracellular/blood environments using 3 mm and 10 microm glutathione in buffer, respectively. The nanocarrier was 9-fold more stable in the extracellular environment. The results suggest selective intracellular degradation of the nanocarrier into components with known body elimination pathways.

Fig. 1

Schematic representation of a monodisperse biodegradable dimeric nanocarrier composed of peptidic backbone irreversibly or reversibly conjugated with one or more targeting ligand/drug either directly or through the distal ends of PEG.

Fig. 2

Synthesis of homodimeric peptide-based PEG nanocarrier^a.

Fig. 3

Synthesis of heterodimeric peptide-based PEG nanocarrier^a.

Fig. 4

Calibration curve of primary amine group of phenylalanine as a function of optical density (570nm). This curve was obtained from quantitative Kaiser chromogenic assay. The concentration of primary amine groups on the 2-, 4- and 6-arm peptidic cores was determined using this chromogenic assay. All measurements were done in triplicate. (R² =0.993)

Fig. 5

MALDI-TOF (m/z) spectrum of crude 6-arm PEGylated peptidic core reaction, showing the heterogeneity of products. The products contain a mixture of conjugation of 2 (7251 Da), 3 (11,082 Da), 4 (14,830 Da), 5 (18,261 Da) and 6 (21,773 Da) copies of m-PEG3.4kDa.

Fig. 6

Gel permeation chromatogram showing separation of labeled ‘Cys-protected fully PEGylated 2-arm peptidic core’ from labeled ‘Cys-protected partially PEGylated 2-arm peptidic core’ and unreacted fluorescein-PEG5kDa using Sephadex G-75 column in 100 mM phosphate buffer pH 7.4±0.2. The fluorescence measurements of each elution volume was detected at Ex = 485 nm; Em = 535 nm corresponding to the fluorescein dye. PEGylation reaction on the 2-arm peptidic core was carried out in two different reaction conditions (a) DMSO: 100 mM phosphate buffer pH 7.4±0.2 (3:7) (▴) (b) DMSO: 100 mM phosphate buffer pH 7.4±0.2 (7:3) (●).

Fig. 7

MALDI-TOF (m/z) spectrum of purified labeled Cys-protected fully PEGylated 2-arm peptidic core. The peak showing molecular weight of 11,099.0 Da confirms attachments of two fluorescein-PEG5kDa to the 2-arm peptidic core.

Fig. 8

HPLC chromatogram of purified Cys-protected PEGylated 2- arm peptidic core (A, C), crude homodimeric peptide-based PEG nanocarrrier (E) and crude homodimeric nanocarrier spiked with purified Cys-protected PEGylated 2- arm peptidic core (B, D, F). Different flow rates have been used to obtain better resolution. Spiking was performed to confirm the formation of the homodimeric nanocarrier and for better visualization.

Fig. 9

MALDI-TOF (m/z) spectrum of purified heterodimeric peptide based PEG nanocarrier doubly labeled with fluorescein and Texas Red, showing a peak at molecular weight of 13,375.6 Da confirming the product.

Fig. 10

Time course release of the dual labeled biodegradable nanocarrier in 3 mM GSH at 37 °C: The heterodimeric nanocarrier was dissolved in 100 mM PB pH 7.4±0.2 in presence of 3 mM reduced GSH. At each time point (0,1,3,5,7,10 and 60 minutes), 1% TFA was added to the sample to stop the reduction reaction. The zero time point is identical to other sample with the exception that it did not contain any GSH. All experiments were performed in triplicate.

Fig. 11

Time course release of the dual labeled biodegradable nanocarrier in 10 μM GSH at 37 °C: The heterodimeric nanocarrier was dissolved in 100 mM PB pH 7.4±0.2 in presence of 10 μM reduced GSH. At each time point (0,15,30,60 and 90 minutes), 1% TFA was added to the sample to stop the reduction reaction by GSH. The zero time point is identical to other sample with the exception that it did not contain any GSH. All experiments were performed in triplicate.