In order to validate size/shape effects of the nanoparticle carrier in their cell internalization characteristics, mediated by PTD peptide, two series of nanoparticle solutions were prepared. The first series included the same spherical structures with increasing loadings of PTD; the second series contained particles having the same amount of PTD but increasing particle length in one dimension (from 10 nm to several μm). The first series was used to probe the effectiveness of PTD on cell internalization while the second series was used to probe the size/shape effect.
We designed and synthesized shell-crosslinked nanoparticles of different sizes and spherical or cylindrical shapes and then performed further functionalization for fluorescent labeling and PTD attachment to afford the two series of materials (). The spherical nanoparticle was assembled from a diblock copolymer (poly(acrylic acid)128
). The short and long cylindrical nanoparticles were assembled from a diblock and a triblock copolymer, PAA98
and poly(acrylic acid)-b
), respectively. All three particles were nominally 30% crosslinked in the shell domain, using standard procedures (28
). The spherical particle had an average diameter of 11 ± 2 nm, as evidenced by transmission electron microscopy (TEM). The short cylinder had an average cross-sectional diameter of 20 ± 2 nm, and an average length of 180 ± 120 nm. The long cylinder had an average cross-sectional diameter of 30 ± 2 nm, and an average length of 970 ± 900 nm (). TEM images () show the morphologies of the three nanostructures.
Block copolymers (PAA128-b-PS40, PAA98-b-PS48 and PAA94-b-PMA103-b-PS28) were micellized by different procedures, to afford spherical and cylindrical nanoparticles of different cross-sectional diameters and lengths.
Dimensions of the nanostructures evaluated.
Size distribution by surface area. The size of the three nanostructures is each separated by an order of magnitude.
Functionalization of the nanostructures with both the fluorescent tag (Alexa Fluor 594 cadaverine) for confocal microscopy and the Tat PTD peptide was achieved by using carbodiimide chemistry. Attachment of the Alexa Fluor 594 to the nanostructures was performed first. To ensure that all final solutions had the same amount of total fluorescence, the solution of spherical particles was then split for further labeling with different amounts of PTD (at levels of 0.5, 1, and 2% consumption of total COOH groups of the PAA128
), while the solutions of cylindrical nanostructures were functionalized at a level that coincided with the 1% labeling of the spheres. Because the Tat PTD has two lysine residues, whose side chain amine could compete with the terminal amine for the activated carboxylic acid on the nanoparticle, ivDde protecting groups were used to cap the lysine residues and were subsequently removed, after the Tat PTD was coupled to the nanoparticle (). A model study on aqueous ivDde removal by Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-ToF) mass spectrometry demonstrated adequate removal under reaction with 2% hydrazine in water (). These reaction conditions were fairly mild to the nanoparticle framework, although some amount of the AlexaFluor 594 cadaverine was cleaved. Therefore, all samples, even the control particles without PTD were treated to these deprotection reaction conditions. Extensive dialysis was used to remove unbound peptide, which previously has been reported to be effective (18
). Both the fluorescent dye and the Tat PTD were loaded successfully within the nanoparticles, as evidenced by UV-Vis spectroscopy (), although it is not clear whether all detected peptide was covalently bound or electrostatically adsorbed onto or absorbed into the nanoparticles, due to their oppositely and highly positively-charged characteristics. For the cylindrical particles, accurate UV-Vis measurement was complicated by light scattering and, therefore, the assumption was made that the coupling yields for all particles were similar (). After functionalization, the morphologies of each nanostructure did not undergo discernible changes, as confirmed by TEM (images not shown).
Scheme 2 Functionalization of the spherical nanoparticle sample was achieved through a three-step synthesis. Alexa Fluor 591 and protected PTD were respectively coupled to the nanoparticle via carbodiimide chemistry. The ivDde protecting groups were removed by (more ...)
UV-Vis spectra of spheres and cylinders labeled with Alexa Fluor 594 cadaverine and PTD peptide.
Light scattering of the long cylinders complicates accurate UV-Vis measurement. A: Long cylinders. B: Short cylinders. C: Spheres.
Because the PTD peptides were conjugated after the micellization and crosslinking and should be present on the particles’ surfaces, the surface areas of the nanostructures determine the number of peptides loaded onto each particle given the total amount of PTD peptide, assuming the particles’ densities are similar (29
). The three nanostructures each represented a unique size distribution, with the greatest percentage surface area for each morphology being separated by an order of magnitude ().
To verify the effectiveness of CPP, spherical particles of the same size loaded with different amounts of CPP (series 1) were incubated with CHO cells at a concentration of 0.68 μM (polymer) at 37 °C and 0 °C for 1 hour. Confocal microscopy imaging of non-fixed, live cells suggests that CPP had a pronounced enhancement in the uptake of the nanoparticles by the CHO cells (). The highest amount of PTD loading, 2.0%, gave nanoparticles that exhibited nearly a 5-fold increase in cell uptake at 37 °C, than the nanoparticles without PTD. Incubation at 0 °C, under otherwise the same conditions, resulted in little cell uptake. The difference in cell uptake at different temperatures can be due to reduced membrane permeability at low temperatures, to the energy-dependency of the uptake process, or to both. An interesting phenomenon we noticed is that the PTD-nanoparticle conjugates were both contained within cellular vesicles and partly spread into the cytoplasm, with exclusion from the nucleus, whereas PTD alone has been reported to accumulate selectively in the cell nucleus (31
). This observation is possibly due to the overall negatively-charged nature of the PTD-particle conjugate or may be due to comparisons between different cell lines.
Confocal images of CHO cells incubated with the spherical nanoparticles having different amounts of PTD for 1 hour. Cell uptake of PTD-sphere conjugates increased by the action of increasing loadings of surface-bound PTD peptide.
To probe the size/shape effect, we loaded the three differently-shaped particles with a fixed amount of PTD (equivalent to 1% of the carboxylates of PAA128
), and incubated CHO cells in their solutions under the same buffer condition as above at 37 °C for 1 hour. It was found that the smaller, spherical particle had higher cell uptake than the larger, cylindrical nanoparticles (). Based on these results, we can speculate on the general mechanisms that govern the transduction of the PTD-functionalized nanomaterials. Many variables determine the uptake rate, including adhesion rate and receptor diffusion kinetics. Freund et al
) and Bao et al
) have modeled these factors and developed a hypothesis involving “wrapping time” of the membrane; the shorter the wrapping time, the faster is the uptake. It has been predicted theoretically by this model that through the competition between thermodynamic driving force and diffusion kinetics, there is an optimum radius for efficient “wrapping”, which is 27−30 nm. Because the early endocytic vesicles are usually less than 100 nm in diameter (6
), most short cylindrical particles (average length = 170 nm) would have to be curved or bent to be internalized, which requires additional energy. Therefore, although the cylindrical particles have higher surface area and hence more membrane-particle interaction (less diffusion), the system is not able to either overcome the overall lack of thermodynamic driving force caused by having to bend the cylinders to “fit” in the endosomes or to create larger endocytic vesicles to contain the cylindrical nanostructures. Another factor, for the higher uptake of the small, spherical particle by the cell at a given time, is the higher particle molar concentration for the spheres, which is the result of the lower aggregation number for the spherical particle, compared with the cylindrical particle. For example, the average short cylinder occupies ca.
80 times as much volume as does the sphere, based on calculations from TEM cross-sectional and longitudinal measurements.
When conjugated with the same amount of PTD peptide, greater amounts of the smaller, spherical nanoparticles were internalized than were the larger, cylindrical nanoparticles. Images were taken after 1 hour of incubation.
Last, we investigated the release of the PTD-nanoparticle conjugates from CHO cells. CHO cells were incubated with 0.68 μM particle solutions for 1 h at 37 °C. Following the incubation period, the nanoparticle-containing solutions were removed, the cells were washed with PBS, and serum free RPMI 1640 media was introduced, followed by further incubation. Fluorescence monitoring of CHO cells immediately after buffer change, and at 2, 4 and 8 h time points revealed that fluorescence intensity decreased over time for spherical nanoparticles labeled with 2%, 1% and 0.5% PTD, suggesting particles being released from the cell (). The rates of release of spherical particles appeared to be dependent on the amount of PTD loaded on the nanoparticle, for which the highest loading (2%) led to the fastest release. This result could suggest that PTD-assisted particle transduction works in a two-way fashion: both entry and exit seem to be facilitated by PTD (35
). The cells that had been incubated with spheres having no PTD did not show apparent decrease of fluorescence over 8 h. Both the long and short cylinders behaved similarly as the spheres without the PTD peptide, showing little uptake and no measurable release. Other PTD-independent processes, such as peptide degradation are possible, and thus more detailed studies on cell uptake and release of differently-shaped nanostructures are needed to reveal a fuller picture of the mechanisms involved.
Release profile of series 1 nanoparticles (spherical in shape; different amounts of PTD peptide) and series 2 nanoparticles (different shapes; each with 1.0% PTD peptide conjugation).