Design and formulation of nanomedicines including liposomes

In this white paper the specific mechanisms by which nanoparticles are designed and formulated for therapeutic purposes and in which NTA has had a significant part to play is discussed.

In this white paper the specific mechanisms by which nanoparticles are designed and formulated for therapeutic purposes and in which NTA has had a significant part to play is discussed.

Nanoparticle Tracking Analysis (NTA) Overview

NTA utilizes the properties of both light scattering and Brownian motion in order to obtain the particle size distribution of samples in liquid suspension. A laser beam is passed through the sample chamber, and the particles in suspension in the path of this beam scatter light in such a manner that they can easily be visualized via a 20x magnification microscope onto which is mounted a camera. The camera, which operates at approximately 30 frames per second (fps), captures a video file of the particles moving under Brownian motion within the field of view of approximately 100 μm x 80 μm x 10 μm (Figure 1).

Figure 1: Schematic of the optical configuration used in NTA.

The movement of the particles is captured on a frame-by-frame basis. The proprietary NTA software simultaneously identifies and tracks the center of each of the observed particles, and determines the average distance moved by each particle in the x and y planes. This value allows the particle diffusion coefficient (Dt) to be determined from which, if the sample temperature T and solvent viscosity η are known, the sphere-equivalent hydrodynamic diameter, d, of the particles can be identified using the Stokes-Einstein equation (Equation 1).


where KB is Boltzmann’s constant.

NTA is not an ensemble technique interrogating a very large number of particles, but rather each particle is sized individually, irrespective of the others. An example of the size distribution profile generated by NTA is shown in Figure 2

Figure 2: An example of the size distribution profile generated by NTA. The modal size for this sample is found to be approximately 70 nm, with larger sized particles also present.

In addition, the particles’ movement is measured within a fixed field of view (approximately 100 μm by 80 μm) illuminated by a beam approximately 10 μm in depth. These figures allow a scattering volume of the sample to be estimated; by measuring concentration of the particles within this field of view and extrapolating to a larger volume it is possible to achieve a concentration estimation in terms of particles per mL for any given size class or an overall total.

Design and formulation of nanomedicines

Villaverde (2011) reviewed the emergence of nanoparticles in translational science and medicine and Banerjee et al. (2010) had focused on the role and biomedical applications that magnetic nanoparticles play in that nanomedicine. Similarly, the impact and biomedical applications of a different specific subset of nanoparticles, nanostructured carbiobeads, has also been reviewed (Stanishevsky et al., 2011).

Wei et al. (2012), in exploring the challenges and opportunities in the advancement of nanomedicines, identified numerous needs including robust and general methods for the accurate characterization of nanoparticle size, shape, and composition as well as particle engineering for maintaining low levels of non-specific cytotoxicity and sufficient stability during storage. They compared NTA and DLS when carrying out size analysis of nanocarriers composed of (a) trimethyl chitosan, (b) 50:50 poly(lactic-co-glycolic acid) (PLGA), and (c) commercial liposomes.

Bai et al. (2012) have recently presented evidence that homogeneous sub-micron particles can influence the growth rate of larger particles upon long-term storage in a temperature-dependent manner with implications for product stability and during which Interferon-beta-1a was thermally stressed at 50 °C for 6 hours and characterized using NTA, microflow digital imaging (MFI), and circular dichroism (CD) spectroscopy.

Following an early assessment of NTA as an emergent technique (Filipe et al., 2010), the role, impact and characterization of NTA has been more recently examined. Cho et al. (2013) discussed state-of-the-art challenges and emerging technologies associated with nanoparticle characterization given the importance they offer as promising tools to enhance target-specific drug delivery and diagnosis. The article provided a critical review of in vitro and in vivo techniques currently used for evaluation of nanoparticles and introduced emerging techniques and models that may be used complementarily and of which NTA was one. In an earlier review of the subject, Poletto had discussed the used of polymeric nanocapsules in the development of nanocosmetics and nanomedicines (Poletto et al., 2011).

Herring et al. (2013), focused on the role of cellular exocytic vesiculation in health, disease, and transfusion medicine as applied to the field of veterinary science assessing the advantages and disadvantages of various assays for the detection of microparticles.

Liposomes, microvesicles and micelles.

Following early work using NTA for the characterization of casein micelles (Thu et al., 2007; Thu et al., 2008) and in the study dispersion of poly(3,4-ethylenedioxythiophene) in organic liquids (Kim et al., 2008), Sorrell and Lyon (2008) studied the deformation controlled assembly of binary microgel thin films.

Despite the frequently low light scattering properties of micellar systems and difficulty in their detection on an individual basis, NTA has been successfully used to characterize such structures (Vakurov et al., 2009) and particularly in the development of drug delivery micellar formulations for controlled release of covalently entrapped doxorubicin (Talelli et al., 2010) and the encapsulation of mithramycin (Capretto et al., 2010; Capretto et al., 2011). This latter study demonstrated that microfluidics was a powerful technology for microfluidic nanoprecipitation-based production of drug loaded polymeric micelles as compared to batch systems since it enabled better control, reproducibility, and homogeneity of the size characteristics of the produced micelles.

In attempting to improve the homogeneity of nano-sized lipid vesicles as drug delivery vehicles made by a constant pressure-controlled extrusion apparatus, Morton et al. (2012) used NTA, DLS and EM to characterize their product’s monodispersity, as did Bhuiyan (2010) in his study of the application of hyperthermia for localized drug release from thermosensitive liposomes.

Wrenn et al. (2012) has used NTA to determine the number of liposomes and their diameter, under the application of ultrasound, in an initial attempt to distinguish mechanisms and quantify the relative contributions of liposome destruction versus diffusion through the bilayer. The overall number of liposomes decreased with ultrasound exposure time, with the most pronounced decrease (nearly 50%) occurring in the first four minutes of ultrasound exposure. This result strongly suggested that at least some vesicle destruction is occurring which was consistent with their prior studies.

Brinkhuis et al. (2012) used NTA to measure the zeta potential of polymersomes, self-assembled from the block copolymer polybutadiene-block-poly(ethylene glycol) in his investigation of the size dependent biodistribution and SPECT imaging of 111In-labeled polymersomes to show that size, much more than for liposomes, will influence the pharmacokinetics, and therefore, long circulating preparations should be well below 100 nm.

Ohlsson et al. (2012) reported on solute transport on the sub-100ms scale across the lipid bilayer membrane of individual proteoliposomes using NTA to check liposome stability and integrity.

Photoactive drug carriers were studied by Reshetov (2012), in which he used NTA to demonstrate that the inclusion of mTHPC into liposomes increases the structural stability of the carriers in serum compared to un-PEGylated liposomes which showed faster kinetics of degradation.

Knowing that membrane curvature and lipid composition regulates important biological processes within a cell, several proteins have been reported to sense and/or induce membrane curvatures, e.g. synaptotagmin-1 and amphiphysin and Morton et al. (2012) have identified a 25-mer peptide, MARCKS-ED, based on the effector domain sequence of the intracellular membrane protein myristoylated alanine-rich C-kinase substrate that can recognize PS with preferences for highly curved vesicles in a sequence specific manner. These studies further contribute to the understanding of how proteins and peptides sense membrane curvature, as well as providing potential probes for membrane shape and lipid composition, NTA being used to monitor vesicle size. Zhu et al. (2012) investigated lipid exchange between membranes and the effects of membrane surface charge, composition, and curvature. Jing et al. (2013) have also studied phase transition-controlled flip-flop in asymmetric lipid membranes by taking advantage of distinct phase transitions in lipid membrane coatings where lipids exchange (flip-flop) between leaflets. The liposomes used in this study were characterized by NTA

Recently, Vader et al. (2013) showed that Taxol®-induced phosphatidylserine exposure and microvesicle formation in red blood cells is mediated by its vehicle Cremophor® EL.


In a study to develop curcumin-loaded lipid-core nanocapsules (C-LNC) in an attempt to improve the antiglioma activity of this polyphenol, visualization of the C-LNC was carried out by NTA (Zanotto-Filho et al., 2012), the data obtained suggesting that the nanoencapsulation of curcumin in LNC is an important strategy to improve its pharmacological efficacy in the treatment of gliomas. More recently, Salehi et al. (2012) have also studied curcumin loaded NIPAAM/VP/PEG-A nanoparticles and studied their physicochemical and chemopreventive properties.

Following early studies in the formation of cholesteric and nematic emulsions (Tixier et al. 2006) NTA was used to follow size changes in nanocapsules for intestinal delivery and enhanced oral bioavailability of tacrolimus, a P-gp substrate (Nyska and Benita 2009) as were other several other nanocarriers (Nasser et al., 2009; Debotton et al., 2010; Sundar et al,. 2010; Smith et al., 2010)

In a case study employing BaTiO3, Pazik et al. (2011) explored the surface functionalization of the metal oxide nanoparticles with biologically active molecules containing phosphonate moieties. Using a battery of techniques including scanning electron microscopy/energy dispersive spectroscopy, pH-metric titration, NMR and IR spectroscopy, DLA, zeta potential, thermogravimetric analysis, radiometric measurements and NTA, they showed that the application of amino phosphonic acids as surface ligands provided nanoparticles with considerable solution stability in an aqueous medium at neutral pH and especially in the presence of electrolytes, thus opening the broad prospect of applications for such nanoparticle dispersions in the domains of nano-optics and nanomagnetism.

Neville et al. (2010) described the fabrication and characterization of biosilicate nanoparticles formed by mimicking the peptides using polyethyleneimine and described, for the first time using TEM and NTA, the characterization of nanoparticles made with tetramethyl orthosilicate to entrap enzymes. This work was explained further in a recent report on the fabrication and characterization of bioactive thiol-silicate nanoparticles (Neville and Millner, 2011). Zu et al. (2012) have also reported the preparation of ultrafine polyethylene-silica composite particles with a core-shell structure using scanning electronic microscope observation and NTA to determine particle sphericity and a mean size of 160 nm respectively.

Researchers investigating the dendritic cell maturation and T cell activation through the application of calcium phosphate nanoparticles encapsulating Toll-like receptor ligands and the antigen hemagglutinin used SEM, DLS, NTA and ultracentrifugation in analyzing size, surface charge, and morphology of the nanoparticles (Sokolova et al., 2010). Similarly, recent developments of a nanoparticulate formulation of retinoic acid that suppresses Th17 cells and up-regulates regulatory T cells employed NTA to measure particle size (Capurso et al. 2010) and the stability of nanometer-sized prodrug (nanoprodrugs) production by a spontaneous emulsification mechanism was confirmed by NTA to be constant, at 120-140 nm in diameter (Lee et al., 2011).

Bhise et al. (2011) have described a novel assay for quantifying the number of plasmids encapsulated by polymer nanoparticles and Capretto et al. (2012) proposed mithramycin encapsulated in polymeric micelles by microfluidic technology as novel therapeutic protocol for beta-thalassemia.

Geng et al. (2012) used NTA to establish that the development and characterization of maleimide-functionalized biopolymer (Mal-PGA-Asp) as an effective targeted drug delivery carrier, synthesized from an amidation reaction between aspartylated PGA (PGA-Asp) and N-(maleimidohexanoyl)-ethylenediamine (NME), led to significantly enhanced cellular uptake of TP13-Mal-PGA-Asp3-Pt in the human hepatoma cell line SMMC-7721 as shown by fluorescence imaging and flow cytometry. NTA was used to show that the biopolymer had an average size 87 ± 28 nm.

Kolluru et al. (2012) also used NTA to develop the optimum formulation of albumin based theranostic nanoparticles as a potential delivery system for tumor targeting showing that both NTA and DLS confirmed that the optimized nanoparticle formulation had a particle size of 125 nm.

The surface coatings of proteins on superparamagnetic iron oxide nanoparticles (SPIONs), that form immediately on contact with a biological milieu, were assessed using a variety of techniques, including NTA, following stabilization of the SPION with citric acid, poly(acrylic acid), or double layer oleic acid (Jedlovszky-Hajdú et al., 2012). SPION have also been exploited by Paquet et al. (2011), when they showed that particle architecture generated a synergistic enhancement of the t2 relaxation in their study of clusters of superparamagnetic iron oxide nanoparticles encapsulated in a hydrogel.

In designing drug delivery vehicles capable of buccal delivery, Mazzarino and her co-workers developed a chitosan-coated nanoparticles loaded with curcumin for mucoadhesive applications by the nanoprecipitation method using different molar masses and concentrations of chitosan and concentrations of triblock surfactant poloxamer (PEO–PPO–PEO) in order to optimize the preparation conditions. DLS studies at different scattering angles and concentrations had shown that the nanoparticles are monodisperse (polydispersity indices were lower than 0.3). The nanoparticle systems were also examined with NTA, and the results were in good agreement with those obtained by DLS.

Colloidal systems showed mean drug content about 460 μg/mL and encapsulation efficiency higher than 99%. When coated with chitosan, these nanoparticles show a great ability to interact with mucin indicating also their suitability for mucoadhesive applications (Mazzarino et al., 2012).

Clementi et al. (2011) determined the hydrodynamic diameter (at 200 nm) and size distribution of PTX-PEG-ALN and of PTX-PEG conjugates by NTA in their work using dendritic poly(ethylene glycol) bearing paclitaxel and alendronate for targeting bone neoplasms.

Chitosan is a natural biodegradable cationic polymer with remarkable potency as a vehicle for drug or vaccine delivery. Zubareva et al. (2013) sought to produce stable nanosized range “Chi-gels” (nanogels, NGs) with different charge and to study the driving forces of complex formation between Chi NGs and proteins or peptides and showed that NGs preferentially formed complexes with oppositely charged molecules, especially peptides, as was demonstrated by gel-electrophoresis, confocal microscopy and HPLC. Complex formation was accompanied by a change in zeta potential and decrease in size as measured by NTA.

Delivery and controlled release

The design and manufacture of micro- and nano-particles capable of releasing, or being triggered to release, a drug cargo in a specific location and at a specific time is one of the biggest opportunities and challenges in nanomedicine. Given knowledge of the size, size distribution profile and number concentration is central to the development and production of such systems, NTA has proved increasingly useful in furnishing this information at all stages through the manufacturing process. Thus, core particle size, the efficiency of addition of functionalized coatings (e.g. antibodies for targeting) and the behavior of such complex, multifunctional structures in biological environments has been the subject of intense study. Here, some examples of such use of NTA are given.

Following earlier work pointing to the potential of NTA in the investigation of complex, multifunctional nanoparticles (Lynch, 2007; Nyska and Benita, 2009), subsequent studies address a wide range of particle types and applications. Pagba and Lane (2010) reported the direct detection of aptamer-thrombin binding via surface-enhanced Raman spectroscopy (SERS) while Ciolkowski et al. (2011) discussed the influence of PAMAM-OH dendrimers on the activity of human erythrocytes ATPases.

The functionalization of nanoparticles was addressed by Park et al. (2011) in their work on the enhancement of surface ligand display on PLGA nanoparticles with amphiphilic ligand conjugates and Kusters et al. (2011) published their work on the functional immobilization of biological membranes in hydrogels. In all these cases, NTA was used to follow particle size during the development stages. Satchi-Fainaro et al. (2011) patented the use of a conjugate of a polymer, an anti-angiogenesis agent and a targeting moiety in the treatment of bone related angiogenesis conditions. ,

Simonsson et al. (2012) presented an amperometric study of content release from individual vesicles in an artificial secretory cell designed with the minimal components required to carry out exocytosis using NTA to measure catechol-filled LUVs at an average diameter of ~200 nm. In fact, using NTA they observed that catechol filled vesicles are larger (mean diameter ≈ 200 nm) than vesicles typically obtained from extrusion though a 100 nm pore sized polycarbonate filter. Hickerson et al. (2012) determined that siRNA–Invivofectamine 2.0 complexes were 100 nm by NTA in his intravital fluorescence imaging of small interfering RNA–mediated gene repression in a dual reporter melanoma xenograft model.

Yandrapu et al. (2012) reported, as an acyclovir model formulation, the development and optimization of thiolated dendrimer as a viable mucoadhesive excipient for the controlled drug delivery while Jensen et al. (2011) elucidated the molecular mechanism of PAMAM-siRNA dendriplex self-assembly in terms of the effect of dendrimer charge density.

Narasimhan et al. (2012) have highlighted an industry perspective of the challenges and technical solutions associated with high-dose monoclonal antibodies via the subcutaneous route.

More recently, increasingly sophisticated structures and applications have been developed and NTA has been used in attempting to determine their structure and optimize the method of production. Chang et al. (2013) reported an aggregation-induced photodynamic therapy enhancement based on linear and nonlinear excited FRET of fluorescent organic nanoparticles, explaining that a binary molecule can self-assemble to form fluorescent organic nanoparticles because of the aggregation-induced emission enhancement property and which subsequently presents an efficient fluorescence resonance energy transfer to generate singlet oxygen under linear and nonlinear light sources. Nanoparticle sizes and sizing partition curves were directly measured using NTA.

Using confocal microscopy to identify the localization of carboxyfluorescein-labeled amylin in RIN-5F cells, Pillay et al. (2013) have developed a direct fluorescent-based technique for cellular localization of amylin. The size of the aggregates that formed on the cell membrane (size range of 130–800 nm) were evaluated using NTA supporting previous findings that amylin was observed to interact with and remain associated to the cell membrane.

Targeted theranostics (combined therapeutic and diagnostic agents) are being developed by inducing clustered nanoconfinement of superparamagnetic iron oxide in biodegradable nanoparticles to enhance transverse relaxivity (Ragheb et al., 2013). Poly(lactide-co-glycolide) nanoparticles were engineered to confine superparamagnetic iron oxide contrast for magnetic resonance imaging while enabling controlled drug delivery and targeting to specific cells. The work showed that clustering of superparamagnetic iron oxide in poly(lactide-co-glycolide), as measured by NTA, did not affect the controlled release of encapsulated drugs such as methotrexate or clodronate and their subsequent pharmacological activity, highlighting the full theranostic capability of the system

In her recent review of currently available drug carriers for oral delivery of peptides and proteins (discussing accomplishments and future perspectives) Reis et al. (2013) pointed out that while effective formulation for peptide and protein delivery through the oral route has always been the critical effort with the advent of biotechnology, stability, enzymatic degradation and ineffective absorption are common difficulties found for conventional dosage forms emphasizing the need for new drug-delivery approaches to circumvent these limitations and enhance effective oral drug delivery.

Design and Formulation

Using DLS and NTA to confirm formulation unimodal size distribution (with polydispersity value <0.1 from DLS) at the nanoemulsion as well as multi-unit pellet system (MUPS) stage, Sangwai et al. (2012) reported a nanoemulsified poorly water-soluble anti-obesity drug Orlistat-embedded MUPS with improved dissolution and pancreatic lipase inhibition. Inclusion of affinity tags greatly facilitated process development for protein antigens, primarily for their recovery from complex mixtures and although generally viewed as supportive of product development, affinity tags may have unintended consequences on protein solubility, susceptibility to aggregation, and immunogenicity.

Khan et al. (2011) employed NTA to establish particle sizes and, importantly, concentrations showing the influence of His-affinity tags on protein expression levels, solubility, secondary structure, thermal denaturation, aggregation and the impact on humoral and cellular immune responses in mice, the results of which suggested that the usefulness of protein tags may be outweighed by their potential impact on structure and function, stressing the need for caution in their use.

Recently, Heljo et al. (2012) explored the stability of rituximab in freeze-dried formulations containing trehalose or melibiose under different relative humidity atmospheres, using NTA to determine the diameter of the nanoparticles was between 50 and 1000 nm. Kasper (2013) has recently comprehensively reviewed multiple aspects of the lyophilization of nucleic acid nanoparticles, including formulation development, stabilization mechanisms, and process monitoring emphasizing the importance of the freezing step. A brief overview on the basic concepts of pDNA and siRNA delivery in gene therapy was given and the need for lyophilized long-term stable formulations was accentuated.

Using NTA to confirm particle diameter, Chernousova et al. (2013) developed a novel genetically active nano-calcium phosphate paste for bone substitution. She showed that especially cationic nanoparticles showed a high transfection efficiency together with a low cytotoxicity. The nanoparticles could be either used in dispersion or added to a calcium phosphate paste for injection into bone defects. Rodrigues et al. (2012) had earlier synthesized and characterized nanocrystalline hydroxyapatite gel and studied its application as scaffold aggregation. Similarly, Stevens et al. (2012) had used NTA in their study of nanosponge formation from organocatalytically synthesized poly(carbonate) copolymers.

Do et al. (2011) undertook the characterization of a lipophilic plasmid DNA condensate formed with a cationic peptide fatty acid conjugate with NTA. Clementi et al. (2011) determined the hydrodynamic diameter (200 nm) and size distribution of PTX-PEG-ALN and of PTX-PEG conjugates by NTA technology in their work using dendritic poly(ethylene glycol) bearing paclitaxel and alendronate for targeting bone neoplasms.

Thermosensitive hydrogels were the subject of another NTA-assisted study by de Graaf et al. (2012) in which they developed a micelle-shedding thermosensitive hydrogel based on poly(N-isopropylacrylamide)-poly(ethylene glycol)-poly(N-isopropylacrylamide) (pNIPAm-PEG-pNIPAm) as sustained release formulation for the delivery of the cytostatic agent paclitaxel (PTX). They showed that, at the highest dose, PTX completely inhibited tumor growth for at least 3 weeks with a single hydrogel injection. This promising concept may find application as a depot formulation for sustained, metronomic dosing of chemotherapeutics.

Mun et al. (2013) have addressed the question of nanoparticle diffusion within non-Newtonian biological and synthetic fluids which though considered essential in designing novel formulations (e.g., nanomedicines for drug delivery, shampoos, lotions, coatings, paints, etc.) is presently poorly defined. Using NTA to visualize nanoparticle diffusion in various media, they reported the diffusion of thiolated and PEGylated silica nanoparticles, characterized by small-angle neutron scattering, in solutions of various water-soluble polymers such as poly(acrylic acid) (PAA), poly(N-vinylpyrrolidone) (PVP), poly(ethylene oxide) (PEO), and hydroxyethylcellulose (HEC) were probed using NTA. The water-soluble polymers retarded the diffusion of thiolated particles in the order PEO > PVP > PAA > HEC whereas for PEGylated silica particles retardation followed the order PAA > PVP = HEC > PEO. They concluded that in the absence of specific interactions with the medium, PEGylated nanoparticles exhibit enhanced mobility compared to their thiolated counterparts despite some increase in their dimensions.

In the development of new imaging agents in cancer therapy, plasmonic gold nanostars which exhibited tuneable plasmons in the near infrared tissue optic window generated intense two-photon photoluminescence capable of in vitro cell labelling and in vivo particle tracking, Yuan recently used the multiparameter analysis capability of an NTA instrument fitted with a zeta potential module to determine the particles' hydrodynamic radius, zeta potential and concentration (Yuan et al. 2012), while Wand and Vo-Dinh (2011) had earlier studied plasmonic coupling interference nanoprobes for nucleic acid detection.

Using NTA to physically characterize their samples, Sunshine et al. (2012) showed that uptake and transfection with polymeric nanoparticles was dependent on polymer end-group structure, but largely independent of nanoparticle physical and chemical properties, while van Galen et al. (2012) demonstrated that the interaction of GAPR-1 with lipid bilayers is regulated by alternative homodimerization. Liling (2008) had earlier employed NTA in his investigations of bio-responsive peptide-inorganic nanomaterials. Troiber et al. (2012) assessed NTA among three other sizing techniques in their comparison of four different particle sizing methods for siRNA polyplex characterization.

Jouffray (2012) described the use of an innovative cross-linked silicone coating in prefilled syringe technology to improve compatibility with biologics given that silicone oil is commonly used as a lubricant coating in prefilled syringes (PFS) and is becoming one of the most highly discussed topics in the PFS market, particularly for developers of highly sensitive biotech drugs. NTA was shown to reveal the presence and formation of sub-visible particles and he showed that the new oil formulation significantly reduced aggregation while retaining lubrication performance using NTA to show the reduction in numbers of 200-1000 nm particles between the novel silicone formulation and baked silicone and conventionally lubricated syringes.

Banerjee et al. 2010 have studied magnetic nanoparticles for radio ablation and magnetic resonance contrast agent development while Smith et al. (2012) have used NTA to show that the change in flux was not a result of a change in size due to aggregation of the haemoglobin at the different pHs tested when confirming that alginate hydrogel has a negative impact on in vitro collagen 1 deposition by fibroblasts.

Finally, Zhuang et al. (2013) have recently reviewed multi-stimuli responsive macromolecules and their assemblies using NTA to characterize micelles under both light scatter and fluorescent modes to explain the origin of employed mechanisms of stimuli responsiveness which may serve as a guideline to inspire future design of multi-stimuli responsive materials.


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