Structure of polymeric nanoparticles encapsulating a drug - pamoic acid ion pair by scanning transmission electron microscopy

Abstract

Drug-delivery systems based on polymeric nanoparticles are useful for improving drug bioavailability and/or delivery of the active ingredient for example directly to the cancerous tumour. The physical and chemical characterization of a functionalized nanoparticle system is required to measure drug loading and dispersion but also to understand and model the rate and extent of drug release to help predict performance. Many techniques can be used, however, difficulties related to structure determination and identifying the precise location of the drug fraction make mathematical prediction complex and in many published examples the final conclusions are based on assumptions regarding an expected structure. Cryogenic scanning transmission electron microscopy imaging in combination with electron energy loss spectroscopy techniques are used here to address this issue and provide a multi-modal approach to the characterisation of a self-assembled polymeric nanoparticle system based upon a polylactic acid - polyethylene glycol (PLA-PEG) block copolymer containing a hydrophobic ion-pair between pamoic acid and an active pharmaceutical ingredient (API). Results indicate a regular dispersion of spherical nanoparticles of 88 ± 9 nm diameter. The particles are shown to have a multi-layer structure consisting of a 25 nm radius hydrophobic core of PLA and pamoic acid-API material with additional enrichment of the pamoic acid-API material within the inner core (that can be off-centre), surrounded by a 9 nm dense PLA-PEG layer all with a low-density PEG surface coating of around 10 nm thickness. This structure suggests that release of the API can only occur by diffusion through or degradation of the dense, 9 nm thick PLA-PEG layer either of which is a process consistent with the previously reported steady release kinetics of the API and counter ion from these nanoparticle formulations. Establishing accurate measures of product structure enables a link to performance by providing appropriate physical parameters for future mathematical modelling of barriers controlling API release in these nanoparticle formulations.

Keywords: Controlled release through diffusion barrier; Cryo-STEM; EELS; PLA-PEG; Polymeric nanoparticles.

Graphical abstract

Fig. 1

(a) Multi-modal approach to investigate the structure of polymeric nanoparticles using cryo-(S)TEM allowing native state analysis of nanoparticles within a rapidly frozen and vitreous suspension, (b) however acquired cryo-(S)TEM images are always a 2D projection of the spherical nanoparticles.

Fig. 2

Cryo-TEM micrograph of polymeric nanoparticles: (a) Low magnification BF-TEM micrograph taken close to focus and (b) close to focus high magnification image (top right micrograph) and the effect of applying under-focus to the same area (bottom right micrograph) in order to increase the particle contrast to allow (c) identification of three individual layers in each nanoparticle (as labelled in the image).

Fig. 3

Electron beam damage observation in cryo-BF-TEM based on imaging the same area with a total electron fluence of (a) 20 e⁻/Ų, (b) 40 e⁻/Ų and (c) 80 e⁻/Ų. Yellow arrows indicate the layer 1 boundary which begins to fade at 40 e⁻/Ų indicating damage to the structure of the nanoparticles. The blue arrows indicate bubble formation, especially at the interface between layer 1 and layer 2, related to severe radiation damage. Based on the observation of the damage propagation, <40 e⁻/Ų (marked in red font) is considered as a safe dose for cryo-TEM imaging. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

(a)–(b) Particle size distribution histograms of radius of layer 1 and thickness of layer 2 measured on the defocused images, respectively. The inserts represent the cumulative mean radius, suggesting that a minimum of 80 particles is required to accurately measure the particle size distribution (since after that number the cumulative radius does not vary significantly). (c) Table with the mean values for layers 1–3. The average diameter of nanoparticles is 88 ± 9 (±SD) nm.

Fig. 5

Cryo-ADF-STEM micrograph of polymeric nanoparticles showing bright central regions in the core of some nanoparticles (green arrows) that might suggest a high density region within an inner core of the nanoparticles. (a)–(b) comparison between under-focus BF-TEM and ADF-STEM of the same region. Yellow curves indicate layer 1 and orange curves indicate layer 2. (c) A dark halo is visible around the particles in the ADF-STEM image (blue arrow and curves) suggesting the presence of layer 3 and it being lower density than the core and the surrounding vitreous ice. d) Application of a high pass filter to the ADF-STEM image removes thickness variation and emphasizes the low electron density of layer 3 and e) inverting the contrast of the high pass filtered ADF-STEM image reveals all three layers of the core-shell-corona nanoparticles. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

(a) Cryo-ADF STEM and cryo-STEM EELS carbon and nitrogen mapping. (b) Cryo-STEM-EEL spectrum acquired over the whole area shows the presence of C, N and O. (c) A linear intensity profile taken from the C–N overlay map and ADF-STEM image indicates that C occurs also between the nanoparticles and N is located approximately in the centre of individual nanoparticles (yellow box in (a)). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

(a) Under-focus BF-TEM image and PCA treated carbon STEM-EELS mapping of the area marked in red. (b) Under-focus BF-TEM, ADF-STEM and C-K EELS mapping of a cropped fragment marked in yellow in (a) and showing carbon presence between the particles, confirming the C-content to layer 3. The corresponding diameter of layer 1 + 2 is identical between cropped under-focus BF-TEM, ADF-STEM and C-K map images (yellow arrows) and the thickness of layer 3 is consistent with earlier measurements of the spacing between nanoparticles by under-focus BF-TEM (blue arrow). Red arrows indicate severe electron beam damage in layer 2 visible in a post-EELS acquisition, ADF-STEM image. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

(a) Cryo-ADF-STEM image of particles and areas where spectra were extracted from – layer 1 (yellow box), layer 2 (orange boxes), layer 3 (blue boxes) and amorphous ice (purple box). (b) Background stripped, as-acquired (faint dots) and smoothed (bold lines) STEM-EELS from layer 1, layer 2 layer 3 and amorphous ice areas in (a). Layer 1 is C rich and also has a clear N peak identifiable. Layer 2 and 3 have progressively less C and relatively more O per layer and less than that of the amorphous ice. (c) Background stripped, as-acquired (faint lines) and smoothed (bold lines) STEM-EELS at the C-K edge reveal that layer 2 and layer 3 have slightly different edge structure to that of layer 1 suggesting a different polymer composition. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

(a) Under-focus BF-TEM of non-standard nanoparticles named as: debris (non-spherical particles, marked as orange arrows) and blank (no visible layer 1, marked as red arrows). Both non-standard particles are highly beam sensitive. (b) Cropped cryo-STEM-EELS mapping of area marked in yellow in (a) suggests that debris and blank particles consist of the same material as layer 2 in the standard particle (i.e., have negligible N content and are highly beam sensitive). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 10

(a) Final multi-modal data-driven model of the polymeric nanoparticles based on (i) ADF-STEM (*high-pass filtered), (ii) phase contrast-TEM and (iii) STEM-EELS analysis. (b) The likely content of each layer is -pamoic acid-API and some PLA material in layer 1 with a core enriched in pamoic acid-API material. Only PEG and PLA in layer 2 and diffuse PEG packing in layer 3.