Characterization of drug delivery vehicles using atomic force microscopy: current status

ABSTRACT

Introduction: The field of nanomedicine, utilizing nano-sized vehicles (nanoparticles and nanofibers) for targeted local drug delivery, has a promising future. This is dependent on the ability to analyze the chemical and physical properties of these drug carriers at the nanoscale and hence atomic force microscopy (AFM), a high-resolution imaging and local force-measurement technique, is ideally suited.

Areas covered: Following a brief introduction to the technique, the review describes how AFM has been used in selected publications from 2015 to 2018 to characterize nanoparticles and nanofibers as drug delivery vehicles. These sections are ordered into areas of increasing AFM complexity: imaging/particle sizing, surface roughness/quantitative analysis of images, and analysis of force curves (to extract nanoindentation and adhesion data).

Expert opinion: AFM imaging/sizing is used extensively for the characterization of nanoparticle and nanofiber drug delivery vehicles, with surface roughness and nanomechanical/adhesion data acquisition being less common. The field is progressing into combining AFM with other techniques, notably SEM, ToF-SIMS, Raman, Confocal, and UV. Current limitations include a 50 nm resolution limit of nanoparticles imaged within live cells and AFM tip-induced activation of cytoskeleton proteins. Following drug release real-time with AFM-spectroscopic techniques and studying drug interactions on cell receptors appear to be on the horizon.     

Monitoring film coalescence from aqueous polymeric dispersions using atomic force microscopy: Surface topographic and nano-adhesion studies

The aim of the investigation was to develop the use of topographic and nano-adhesion atomic force microscopy(AFM) studies as a means of monitoring the coalescence of latex particles within films produced from a pharmaceutically relevant aqueous dispersion(Eudragit~?NE30 D). Films were prepared via spin coating and analysed using AFM, initially via tapping mode for topographic assessment followed by force-distance measurements which allowed assessment of site-specific adhesion. The results showed that colloidal particles were clearly observed topographically in freshly prepared samples, with coalescence detected on curing via the disappearance of discernible surface features and a decrease in roughness indices. The effects of temperature and humidity on film curing were also studied, with the former having the most pronounced effect. AFM force measurements showed that the variation in adhesive force reduced with increasing curing time, suggesting a novel method of quantifying the rate of film formation upon curing. It was concluded that the AFM methods outlined in this study may be used as a means of qualitatively and quantitatively monitoring the curing of pharmaceutical films as a function of time and other variables, thereby facilitating rational design of curing protocols.     

The use of atomic force microscopy to study the conditioning of micronised budesonide

Patent literature describes “conditioning” techniques which employ organic vapours to recrystallise amorphous regions in micronised particles, with the aim of improving their processability and physico-chemical stability. This report describes a preliminary study investigating the efficacy of PhaseImaging™ atomic force microscopy (AFM) for the investigation of such processes. AFM phase images demonstrated variation in mechanical properties across the surface of milled budesonide particles, which diminished upon exposure to ethanol vapour. No variation was seen in phase images of unmilled budesonide. Dynamic vapour sorption confirmed the presence amorphous material in the milled sample and its subsequent recrystallisation following exposure to ethanol vapour under the same conditions as those used in the AFM experiment. It was therefore hypothesised that variation in the phase images indicated the presence of amorphous regions which were subsequently conditioned. PhaseImaging™ AFM may therefore be a useful method for the study of conditioning techniques, enabling the efficacy and kinetics of the process to be observed.     

Structural evaluation of probucol nanoparticles in water by atomic force microscopy

Structural evaluation of probucol nanoparticles coground with polyvinylpyrrolidone K17 and sodium dodecyl sulfate for 90 min was performed by solid-state nuclear magnetic resonance (NMR) spectroscopy and atomic force microscopy (AFM) with force-distance curve analysis. The results of solid-state NMR indicated that the cogrinding changed crystalline probucol to amorphous form. The number-averaged mean heights of probucol particles in the ground mixture (GM) suspension were determined by AFM to be 6 and 15 nm for freshly prepared and 24h-stored samples, respectively. Nucleation and the subsequent crystal growth might have occurred after the GM was dispersed in water. The presence of probucol nanocrystals and agglomeration of the primary probucol nanoparticles were recognized by AFM force-distance curve analysis. AFM could be a promising tool to evaluate the structure of nanoparticles as well as their agglomeration behavior in aqueous media.     

Synthesis of poly(sebacic anhydride)-indomethacin controlled release composites via supercritical carbon dioxide assisted impregnation

The characteristics of various pharmaceutical dosage forms are influenced by surface properties such as the friction behavior. For example, die wall friction is a key issue in developing a solid dosage form. However, the friction properties are not completely understood mainly because of the lack of fundamental measurements. Herein, the friction behavior of pharmaceutical materials was investigated and compared with their adhesion behavior using atomic force microscopy. The sliding speed causes significant variations in the frictional force. Compared with other materials, lubricant materials showed less distinct differences in friction tests than in adhesion tests, indicating the dependence of the lubricant efficiency on the stress state. The three parameters obtained from the modified Amonton's law, i.e., absolute frictional force, friction coefficient and residual force, showed consistent trends. Overall, the friction behavior was not a direct reflection of the adhesion forces. The intrinsic friction behavior of a single pharmaceutical particle can be quantified using atomic force microscopy.     

An atomic force microscopy investigation of bioadhesive polymer adsorption onto human buccal cells

Atomic force microscopy (AFM) was used to examine the buccal cell surface in order to image the presence of adsorbed bioadhesive polymers identified from previous work. Isotonic saline solution (5 ml) containing either polycarbophil (pH 7.6), chitosan (pH 4.5) or hydroxypropyl methylcellulose (pH 7.6) (0.5% w/v) was exposed to freshly collected buccal cells (ca. 48×10⁴ cells/test) for 15 min at 30°C. The cells were then rinsed with a small volume of double distilled water, allowed to air-dry on a freshy cleaved mica surface and imaged using contact mode AFM. Untreated cells showed relatively smooth surface characteristics, with many small ‘crater-like’ pits and indentations spread over cell surfaces. Cells that had been treated with all the investigated polymers appeared to have lost the crater and indentation characteristic and gained a higher surface roughness. These results suggest that polymer chains had adsorbed onto the cell surfaces. Quantitative image analysis of cell topography showed significant increases (P<0.05) in arithmetic roughness average (R_a) for all the investigated polymer treated cells surfaces with respect to untreated control specimens. The changes in surface topography indicate the presence of adsorbed polymer, confirming previous work. This study demonstrates the suitability of AFM as a powerful and sensitive technique for detecting and imaging bioadhesive polymers present on mucosal cell surfaces.     

Effect of colistin exposure and growth phase on the surface properties of live Acinetobacter baumannii cells examined by atomic force microscopy

The diminishing antimicrobial development pipeline has forced the revival of colistin as a last line of defence against infections caused by multidrug-resistant Gram-negative ‘superbugs’ such as Acinetobacter baumannii. The complete loss of lipopolysaccharide (LPS) mediates colistin resistance in some A. baumannii strains. Atomic force microscopy was used to examine the surface properties of colistin-susceptible and -resistant A. baumannii strains at mid-logarithmic and stationary growth phases in liquid and in response to colistin treatment. The contribution of LPS to surface properties was investigated using A. baumannii strains constructed with and without the lpxA gene. Bacterial spring constant measurements revealed that colistin-susceptible cells were significantly stiffer than colistin-resistant cells at both growth phases (P < 0.01), whilst colistin treatment at high concentrations (32 mg/L) resulted in more rigid surfaces for both phenotypes. Multiple, large adhesive peaks frequently noted in force curves captured on colistin-susceptible cells were not evident for colistin-resistant cells. Adhesion events were markedly reduced following colistin exposure. The cell membranes of strains of both phenotypes remained intact following colistin treatment, although fine topographical details were illustrated. These studies, conducted for the first time on live A. baumannii cells in liquid, have contributed to our understanding of the action of colistin in this problematic pathogen.     

Surface characterization by atomic force microscopy of sterilized PLGA microspheres

Atomic force microscopy (AFM) is recognized a suitable and powerful technique for surface and morphological analysis. Even if until now this technique has not been frequently used in the pharmaceutical field, it can contribute to an accurate morphologic characterization of microspheres and nanospheres. In this work, atomic force microscopy has been used to perform the surface characterization of sterilized microspheres. The aim is to investigate the morphologic modifications induced by gamma irradiation on poly(lactide-co-glycolide) microspheres loaded with ovalbumin and to compare the results obtained by AFM to those obtained by scanning electron microscopy (SEM). The results obtained show that, with respect to SEM, AFM can give some additional information regarding the modifications induced by γ-irradiation on microspheres surface morphology. The significant changes in surface roughness after irradiation are indicative of damage due to γ-irradiation. The unchanged surface roughness values calculated for microspheres containing PEG in their matrix, suggest that this polymer exerts a protective effect towards γ-irradiation.     

乙酰胆碱对乙酰胆碱酯酶立体结构影响的初步原子力显微观察

目的　为探索乙酰胆碱酯酶 (AChE)活性中心狭隙与其快速水解功能的适应原理 ,研究乙酰胆碱 (ACh)对AChE立体结构的影响。方法　在云母表面重组磷脂膜 ,将AChE重组到磷脂膜上 ,用原子力显微技术观察。结果　天然AChE表面平滑 ,边界清晰 ,中间突出 ,略呈椭圆球体 ,蛋白颗粒长径为(6 2± 2 5 )nm ,短径为 (5 3± 13)nm ,高度为 (6± 2 )nm ;反应后酶蛋白表面变得粗糙 ,形态不甚规则 ,蛋白颗粒中心出现近圆形孔洞。结论　ACh对AChE立体结构具有明显影响。     

Applications of biosensing atomic force microscopy in monitoring drug and nanoparticle delivery

Introduction: The therapeutic effects of medicinal drugs not only depend on their properties, but also on effective transport to the target receptor. Here we highlight recent developments in this discipline and show applications of atomic force microscopy (AFM) that enable us to track the effects of drugs and the effectiveness of nanoparticle delivery at the single molecule level.

Areas covered: Physiological AFM imaging enables visualization of topographical changes to cells as a result of drug exposure and allows observation of cellular responses that yield morphological changes. When we upgrade the regular measuring tip to a molecular biosensor, it enables investigation of functional changes at the molecular level via single molecule force spectroscopy.

Expert opinion: Biosensing AFM techniques have generated powerful tools to monitor drug delivery in (living) cells. While technical developments in actual AFM methods have simplified measurements at relevant physiological conditions, understanding both the biological and technical background is still a crucial factor. However, due to its potential impact, we expect the number of application-based biosensing AFM techniques to further increase in the near future.     

Towards screening of inhalation formulations: measuring interactions with atomic force microscopy

This review charts the progress of atomic force microscopy (AFM) to investigate particle interactions relevant to the performance of inhalers. AFM provides a unique opportunity to examine and quantify single particle behaviour of powdered drugs and excipients in a variety of environmental conditions. An introduction to AFM and particle interactions is given. Comparative experiments that rank adhesion between materials, and quantitative experiments that lead to the measurement of properties such as the work of adhesion and surface energy, are reviewed. The AFM has been widely used to investigate the effects of relative humidity and surface roughness on particle adhesion; these experiments are also reviewed. In the final section, the potential of this approach to screen formulations is discussed.     

Towards crystal engineering via simulated pulmonary surfactant monolayers to optimise inhaled drug delivery

Purpose

To generate theophylline monohydrate crystals underneath Langmuir monolayers composed of material expressed at the alveolar air-liquid interface. Such monolayers can act as nucleation sites to direct crystallisation. The approach offers a novel route to rationally engineer therapeutic crystals and thereby optimise inhaled drug delivery.

Methods

Langmuir monolayers consisting of either dipalmitoylphosphatidylcholine (DPPC) or a surfactant mix reflecting pulmonary surfactant were supported on an aqueous theophylline (5.7 mg/ml) subphase. The monolayers were compressed to surface pressures reflecting inhalation and exhalation (i.e. 5 mN m⁻¹ or 55 mN m⁻¹) with a period of 16 h to allow crystallisation. Analysis involved scanning electron microscopy (SEM), atomic force microscopy (AFM) and powder X-ray diffraction (PXRD).

Results

Condensed isotherms were acquired, which signified surfactant-theophylline interaction. Theophylline monohydrate crystals were obtained and exhibited needle-like morphology. SEM and AFM data highlighted regions of roughened growth along with smooth, stepwise growth on the same crystal face. The surfactant monolayers appeared to influence crystal morphology over time.

Conclusions

The data indicate a favourable interaction between each species. The principal mechanism of interaction is thought to be an ion-dipole association. This approach may be applied to generate material with improved complementarity with pulmonary surfactant thus enhancing the interaction between inhaled drug particles and internal lung surfaces.     

Nanoscale monitoring of drug actions on cell membrane using atomic force microscopy

Knowledge of the nanoscale changes that take place in individual cells in response to a drug is useful for understanding the drug action. However, due to the lack of adequate techniques, such knowledge was scarce until the advent of atomic force microscopy (AFM), which is a multifunctional tool for investigating cellular behavior with nanometer resolution under near-physiological conditions. In the past decade, researchers have applied AFM to monitor the morphological and mechanical dynamics of individual cells following drug stimulation, yielding considerable novel insight into how the drug molecules affect an individual cell at the nanoscale. In this article we summarize the representative applications of AFM in characterization of drug actions on cell membrane, including topographic imaging, elasticity measurements, molecular interaction quantification, native membrane protein imaging and manipulation, etc. The challenges that are hampering the further development of AFM for studies of cellular activities are aslo discussed.     

The effects of age on the response to caffeine

Twelve healthy subjects, six young and six elderly, of either sex, took part in this two-period crossover study. In each session, a dose of trial drug — either 200 mg caffeine or a matching placebo — was given orally at 0900 hours. A battery of psychomotor tests and visual analogue scales was administered before treatment and at 1, 2 and 3 h post-treatment. The objective tests showed a significant increase in tapping rate in the young, while the elderly showed improved attention, faster choice-reaction time, and greater body sway on caffeine. The visual analogue scales showed that the young subjects felt more alert, calmer, more interested, and steadier on caffeine, while no significant changes were seen in the elderly. These results show that caffeine produces changes predominantly in the direction of improved performance and feeling of well-being, and suggest that the elderly are more sensitive to the objective effects of the drug, while reporting less subjective effect than the young.     

基于原子力显微镜的损伤星形胶质细胞弹性模量变化的研究

目的 探讨星形胶质细胞受到损伤刺激后弹性模量的变化。方法 分离、 提取新生 2 d SD 大鼠星形胶质细胞, 通过胶质纤维酸性蛋白 （GFAP） 抗体免疫荧光染色对其进行鉴定。实验分为对照组和损伤组, 损伤组为应用细胞损伤仪损伤后 6 h 的星形胶质细胞, 对照组不予损伤。应用原子力显微镜在液相下测试各组细胞的弹性模量, 并对两组结果进行比较、 分析。结果 大鼠星形胶质细胞纯化率达 95%以上。损伤后 6 h 的星形胶质细胞形态紊乱,部分细胞胞体可见肿胀。获得了星形胶质细胞的力学地形图和力压痕曲线。损伤组星形胶质细胞弹性模量较对照组显著增加［（1 689±693） Pa vs.（724±283） Pa, P＜0.01］。结论 损伤刺激会造成星形胶质细胞弹性模量增大, 为进一步从细胞水平了解创伤性脑损伤后的颅内物理微环境提供理论基础。     

Study of a pingyangmycin delivery system: Zein/Zein-SAIB in situ gels

Venous malformations (VM) are common vascular abnormality, and their management remains difficult. Pingyangmycin hydrochloride (PYM) is a useful sclerosant to treat venous malformations. This study was aimed to evaluate the effect of a new drug delivery system, PYM-loaded Zein/Zein-sucrose acetate isobutyrate (SAIB) in situ gels, in gelling and extending the local release of PYM. It was demonstrated that in vitro and in vivo release of PYM from the in situ gels could be extended up to 7 and 4 days, respectively. SAIB could significantly cut down the initial burst of PYM from the in situ gels (P<0.05). The possible gel forming and drug release mechanisms were described according to the morphology analysis by atomic force microscopy (AFM), optical microscopy and SEM. The gel forming efficacy and the viscosity of in situ gel solutions were satisfying.     

Atomic force microscopy: a tool to study the structure,dynamics and stability of liposomal drug delivery systems

Much work has been done during the past few decades to develop effective drug delivery systems (DDS), many of which are based on nanotechnology science. Liposomes are the most attractive lipid vesicles for drug delivery. The multifunctional properties of liposomes have a key role in modifying the bioavailability profile of a therapeutic agent. Different analytical techniques can be used to describe liposomes, not least applied scanning probe microscopy (SPM) techniques. Atomic force microscopy (AFM) seems to be one of the most effectively applied SPM techniques. This review article outlines the applications of AFM in evaluating the physical characteristics and stability of liposomal DDSs. Other well-known microscopy techniques used in evaluating liposome physical characteristics are also mentioned, and the contribution of AFM to evaluating liposomal stability is discussed. Among the advantages of AFM in examining the physicochemical properties of liposomal DDSs is its ability to provide morphological and metrology information on liposome properties. AFM thus appears to be a promising tool in technological characterization of liposomal DDSs.     

Parameters influencing polymer particle layering of the dry coating process

The dry coating process is an emerging coating technology using neither organic solvents nor water. In contrast to liquid-borne coatings, coating material application and film formation are divided into two phases, the coating phase where the powdery coating material is applied together with the liquid plasticizer, and the curing phase. In this study the coating phase was characterized with respect to the forces acting between the polymer particles during material application. Atomic force microscopy was conducted measuring the interparticle forces which were related to the coating efficiency. The influence of different liquid additives on the interparticle forces and the coating efficiency were evaluated. HPMCAS was used as enteric resistant polymer, triethylcitrate (TEC), Myvacet (diacetylated monoglyceride) and a mixture of both as liquid additives. Interparticle forces were found to be similar when using TEC or a mixture of TEC and Myvacet. In contrast, interparticle forces were higher when using solely Myvacet. This is attributed to the fact that Myvacet does not penetrate into the polymer without TEC which is acting as a penetration enhancer. As Myvacet remains predominantly on the particle surface, capillary forces act between the particles explaining high interparticle forces. The highest interparticle force determined by AFM is in accordance to the highest coating efficiency which has been found for the corresponding coating formulation containing HPMCAS and Myvacet. Consequently, it is demonstrated that the ability of the liquid to remain on the surface of the polymer and to build up capillary forces is crucial for the material application.     

Application of Raman microscopy and band-target entropy minimization to identify minor components in model pharmaceutical tablets

The aim of the current study is to establish a useful analytical technique to detect and identify minor components of pharmaceutical drug tablets using Raman microscopy and advanced multivariate data analysis method, namely band-target entropy minimization (BTEM). Model pharmaceutical tablets comprising four components with varying proportions were prepared with a custom press tooling after blending. One of the components, magnesium stearate, was made as a minor component, with weight percentages of 2%, 1%, 0.5%, and 0.2% in four model tablets. Raman point-by-point mapping was performed on an area of 200 microm x 200 microm using a near infrared laser source and a 50x objective lens with a step size of 5 microm in both the x and y directions. Advanced multivariate analysis, BTEM, was then performed on the Raman mapping data to recover all observable pure component spectra. BTEM was successfully applied to recover the pure component spectrum of magnesium stearate, which was present as a minor component (as low as 0.2 wt%) in the prepared tablet. The success of BTEM in identifying minor chemical species offers a new analytical technology for detecting impurities or any other minor components in pharmaceutical tablets.     

Effects of N-cyclopentyladenosine and caffeine on sleep regulation in the rat

To study the role of adenosine in sleep regulation, the adenosine A₁ receptor agonist N⁶-cyclopentyladenosine (CPA) and the antagonist caffeine were administered to rats. Intraperitoneal (i.p.) CPA 1 mg/kg but not 0.1 mg/kg, suppressed rapid-eye-movement (REM) sleep and enhanced electroencephalographic (EEG) slow-wave activity (power density 0.75–4.0 Hz) in non-REM sleep. The latter effect was remarkably similar to the response to 6-h sleep deprivation. The effects persisted when CPA-induced hypothermia was prevented. Caffeine (10 and 15 mg/kg i.p.) elicited a dose-dependent increase in waking followed by a prolonged increase of slow-wave activity in non-REM sleep. The combination of caffeine (15 mg/kg) and sleep deprivation caused less increase in slow-wave activity than sleep deprivation alone, indicating that caffeine may reduce the buildup of sleep pressure during waking. The results are consistent with the involvement of adenosine in the regulation of non-REM sleep.