Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Liquid colloidal systems are used for several pharmaceutical drug delivery systems. For inhalation drug delivery, a common colloidal system is the suspension pressurized metered dose inhaler (pMDI). Particle interactions present within the pMDI play a vital role in formulation physical stability, storage, and drug delivery uniformity. In this manuscript, inter-particle forces between porous lipid-based particles (~2 µm optical mean particle diameter) in a model propellant (2H,3H-perfluoropentane) were evaluated at RT to convey the functionality of and possible errors associated with the presented procedure.

Figure 3 shows two representative colloidal probes prepared using the lipid-based inhalable particles that can be used for colloidal probe nanoscopy. It is important that a single colloidal particle is affixed to the apex of the cantilever such that it is the most prominent feature and will be the first point of contact during the measurement. This ensures that the interactions measured are solely due to the colloid particle. Attaching multiple particles or particle agglomerates can produce erroneous results (Figure 4) due to multiple cantilever deflections caused by both particles simultaneously sensing the same single particle present on the substrate. Using properly prepared colloidal probes, topographical images of a particle substrate such as those shown in Figure 5 can be achieved in a liquid medium.

Topography scans using a colloidal probe will be less defined than those obtained using a sharpened conical tip; however, in CPN, the main purpose of a topographical scan is to locate a particle on the substrate that can be used to evaluate the inter-particle interactions. Figure 6 conveys several force curves one may encounter when conducting colloidal probe measurements in a liquid medium. Liquid measurements contain more sources of error during measurement and one should be aware of all sources to appropriately minimize their effect on the accuracy of the measurement (Figure 6A).

The rapid and sharp peaks evident in the force curve in Figure 6B are indicative of a sudden disturbance to the system during the measurement. This can be attributed to AFM instrument movement or a sudden noise in the background (ex. door slamming, sneezing) that results in a short period of instantaneous and rapid destabilization. In Figure 6C the fluctuation of the baseline in the approach and retraction of the cantilever suggest a problem with the liquid medium. This may occur if the liquid cell is not adequately filled allowing the evaporation of the medium to have a large impact on the stability of the system and measurement. An alternative source of this instability can be from improper equilibration of the cantilever in the liquid medium prior to analysis. The cantilever is sensitive to changes in temperature and actions such as ‘topping off’ the liquid cell require adequate re-equilibration time. Figure 6D depicts a baseline shift during the approach and retract cycle. This clean shift is non-existent during instantaneous force measurements, but is more apparent in dwell force measurements. This drift is an effect of cantilever thermal drift, which can occur for several reasons including: slow evaporation of the liquid medium leading to a change in the temperature of the medium, using a medium that is still equilibrating to the surrounding temperature, or conducting the measurement in an environment that is not ideally controlled. Minimal steady shifts in the temperature of the liquid medium during the measurement produce such drifts. This type of measurement drift is difficult to control for high evaporating liquids, unless a closed liquid cell is used during measurement; however, most AFM analysis software can correct such drifts.

After all controllable sources of error have been mitigated and the system is appropriately equilibrated, adhesion mapping can be employed to obtain a large statistical body of data across a determined size of the sample. Force mapping can be used independently or in conjunction with topographical scanning to evaluate the effect of topography on particle adhesive forces (Figure 7). Force mapping will provide two major graphs of interest: a topographical map of the sample based on the height at which the cantilever contacts the substrate (Figure 7A) and an adhesion map conveying the max pull force of each individual force curve (Figure 7B). The graph in Figure 7B can also be used to obtain a numerical average and standard deviation of adhesion and snap-in forces, as well as separation energies across the entire sample. These raw data graphs can be viewed as three-dimensional representations of the topography or spread of adhesion measurements across the sample (Figure 7C/D) and overlaying them will produce a three-dimensional illustration of the distribution of the adhesion forces as a function of the topography (Figure 7E). This type of data provides critical understanding of the forces present between colloids and how the surface of the colloids further effect interactions.

Additionally dwell force measurements can be used to evaluate the effect of contact mechanics and length of contact on the adhesive forces. Solid lipid particles were used to convey the effect of the dwell on the measured adhesive forces (Figure 8). Figure 8 indicates that adhesive forces increase as a function of time using indentation dwell, while they plateau using deflection dwell. This trend becomes more obvious at longer dwell times (180 sec).

Figure 1. Depiction of the method used to produce colloidal probes for colloidal probe nanoscopy. (A) AFM cantilever, attached to a custom designed 45° cantilever holder; (B) Epoxy/Particle slide is affixed to a secondary holder that is slid onto the microscope lens; (C) The cantilever is slowly raised to acquire epoxy and a particle.

Figure 2. The sensitivity of the cantilever is the slope of the contact region of a deflection vs. z-distance curve.

Figure 3. Properly prepared Colloidal Probes that can be used to conduct the colloidal probe measurements.

Figure 4. The use of a colloidal probe that has multiple particles affixed may result in the erroneous duplication of a single particle present on the substrate during the topographical scanning of the substrate.

Figure 5. Topography scans obtained using a properly prepared colloidal probe. (A) A large scan revealing multiple particles of interest; (B) a more focused scan revealing one major particle of interest; (C) a scan focused on the surface of a single particle.

Figure 6. Force curves obtained with various limitations that one must be aware of. (A) Example of a good force curve; (B) force curve showing a disturbance either by movement of the AFM or by noise present during the measurement; (C) fluctuation due to un-equilibrated cantilever can lead to unstable approach/retract; (D) thermal drift present during a measurement, existent because of slow evaporation leading to cooling of the medium or unstable environmental control.

Figure 7. Adhesion force mapping that can be obtained using colloidal probe nanoscopy. (A) Topographical distribution of the sample surface; (B) distribution of max adhesion force across the sample. (C/D) 3-dimensional representations of the graphs shown in a and b respectively; (E) overlay of the topography and adhesion forces producing a single three-dimensional illustration of the adhesion forces as a function of topography.

Figure 8. Adhesion forces measured as a function of dwell time using two varying dwell measurements (n=30), (◊) indentation (□) deflection; * indicates a significant difference between values at the specific time point using a two-tailed T-test with 95% confidence (P < 0.05).