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Nano-Encapsulated Intranasal Vaccine Against S. Pneumoniae with Ultrasonics

Nanoparticle-based drug carriers are an evolving technology, which allows to formulate vaccine delivery systems with a high protection rate against various diseases. Ultrasonic emulsification and encapsulation is a highly efficient method to prepare loaded nano-structured drug carriers such as nanoparticles, solid-lipid nanoparticles, polymeric drug carriers, and liposomes.

Advantage of Nanoparticle-Encapsulated S. pneumoniae Vaccines

The ultrasonicator UP200St in a stirred vessel for emulsification of reactantsMott et al. (2013) determined the efficacy of intranasal delivery of a 234 ± 87.5nm poly lactic-co-glycolic acid nanoparticle vaccine construct in establishing protection against experimental respiratory pneumococcal infection. Nanoparticles encapsulating heat-killed Streptococcus pneumoniae (NP-HKSP) were retained in the lungs 11 days following nasal administration compared to empty NP. Immunization with NP-HKSP produced significant resistance against S. pneumoniae infection compared to administration of HKSP alone. Increased protection correlated with a significant increase in antigen-specific Th1-associated IFN-c cytokine response by pulmonary lymphocytes. This study establishes the efficacy of NP-based technology as a non-invasive and targeted approach for nasal-pulmonary immunization against pulmonary infections.

Protocol of Ultrasonic Nanoparticle Preparation

Ultrasonic Lysis

1×106 nanoparticles encapsulating heat-killed Streptococcus pneumoniae (NP-HKSP) were lysed by sonication in 200µl of phosphate-buffered saline (PBS), and 70 mg of poly lactic-co-glycolic acid (PLGA) was dissolved in 1 ml of ethyl acetate. These two solutions were mixed and vortexed at maximum speed for 1 min to form primary water-in-oil emulsion.

Ultrasonic Encapsulation

Double Emulsion Method: The primary emulsion was then mixed with 3 ml of 1 % polyvinyl alcohol (PVA) solution. This solution was sonicated using an ultrasonic processor UP200H (Hielscher Ultrasonics GmbH, Germany) at 40 % amplitude for 2 min on continuous mode (100% cycle), in a clean glass vial immersed in ice for heat dissipation, to prepare HKSP encapsulating PLGA nanoparticles. The solution was further diluted to 20ml with autoclaved water (0.22µ filter sterilized) and stirred for 1 h at room temperature under mild vacuum to evaporate ethyl acetate. The solution was then centrifuged to collect NPs, and this process was repeated twice to remove excess PVA. The nanoparticle pellet was resuspended in 500µl of autoclaved water and freeze-dried. The final nanoparticles were stored at -20°C until further use.

Partcle size measurement of ultrasonically prepared heat-killed Streptococcus pneumoniae-loaded nanoparticles.

Particle size of heat-killed Streptococcus pneumoniae-encapsulated PLGA nanoparticles. The particle size of an aqueous suspension of nanoparticles measured by dynamic light scattering shows the average size and the Gaussian distribution of particles in the batch.
Source: Mott et al.: Intranasal delivery of nanoparticle-based vaccine increases protection against S. pneumoniae. J Nanopart Res (2013) 15:1646.

Ultrasonic processor UIP2000hdT (2kW) with stirred batch reactor

Ultrasonic homogenizer UIP2000hdT (2kW) with continuously stirred batch reactor

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Ultrasonic Processors for Pharmaceutical Formulations

Hielscher Ultrasonic is long-time experienced in the design, manufacturing, distribution and service of high-performance ultrasonic homogenisers for the pharmaceutical and food industry.
The preparation of high-quality liposomes, solid lipid nanoparticles, polymeric nanoparticles and cyclodextrin complexes are processes, which Hielscher ultrasonic systems are used with high reliability and superior quality output. Hielscher ultrasonicators allow for precise control over all process parameters, such as amplitude, temperature, pressure and sonication energy. The intelligent software automatically protocols all sonication parameters (time, date, amplitude, net energy, total energy, temperature, pressure) on the built-in SD-card.

The Advantage of Ultrasonic PLGA Nanoparticle Preparation

  • High performance emulsification
  • Exact control over particle size and load
  • High load of active substances
  • Exact control over process parameters
  • Fast Process
  • Non-thermal, precise temp control
  • Linear Scalability
  • Reproducibility
  • Process standardisation / GMP
  • Autoclavable probes and reactors
  • CIP / SIP

The table below gives you an indication of the approximate processing capacity of our ultrasonicators:

Batch Volume Flow Rate Recommended Devices
1 to 500mL 10 to 200mL/min UP100H
10 to 2000mL 20 to 400mL/min UP200Ht, UP400St
0.1 to 20L 0.2 to 4L/min UIP2000hdT
10 to 100L 2 to 10L/min UIP4000hdT
n.a. 10 to 100L/min UIP16000
n.a. larger cluster of UIP16000

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Hielscher Ultrasonics manufactures high-performance ultrasonic homogenizers for dispersion, emulsification and cell extraction.

High-power ultrasonic homogenizers from lab to pilot and industrial scale.



Facts Worth Knowing

Nano-Structured Drug Carriers

Nano-sized drug carriers such as nano-emulsions, liposomes, solid-lipid nanoparticles, polymeric nanoparticles, and nano-structured lipid carriers are used to formulate pharmaceuticals with enhanced functionalities such as improved bioavailability, increased biocompatibility, targeted delivery, favorable blood half-life, and very low or no toxicity to healthy tissues. Ultrasonication is a highly efficient technique to formulate various forms of nanotherapeutics. Read more about ultrasonic applications in the pharmaceutical production!

Liposomes

A liposome is a spherically shaped vesicle having at least one lipid bilayer, which encapsulates the core of hydrophobic substances. Both, size as well as the hydrophobic and hydrophilic character turn liposomes into potent drug delivery systems, e.g. liposomal vitamin C. Liposome characteristics are substantially influenced by lipid composition, surface charge, size, and preparation technique. Click here to learn more about the ultrasonic preparation of liposomes!

Nano-Emulsions

Nanoemulsions or submicron emulsions are emulsions with a droplet size between 20-200nm and a narrow droplet distribution. The nano-sized droplets offer several advantages for oral administration as well as for topical / transdermal delivery of pharmaceutical and bioactive substances, e.g. CBD nanoemulsions. The nano-sized droplets with the ability to efficiently dissolve lipophilic drugs as well as the enhanced absorption rate make nano-emulsions a frequently used administration form for a high bioavailability. Nano-emulsified formulations can also used for an extended release of lipophilic or hydrophilic drugs.
Read more about the ultrasonic production of nano-emulsions!

Solid-Lipid Nanoparticles

A solid lipid nanoparticle (SLN) is a spherical nanoparticle with a mean diameter between 10 and 1000 nanometers. Solid lipid nanoparticles have a solid lipid core matrix in which lipophilic molecules (active substances) can be solubilized so that the nanoparticle acts as a drug carrier. The lipid core is stabilized by an emulsifying agent or surfactant. With applications for parenteral and oral administration as well as ocular, pulmonary and topical drug delivery, solid-lipid nanoparticles are used to enhance treatment efficacy and to reduce systemic side effects.
Read more about the ultrasonically-assisted synthesis of solid-lipid nanoparticles!

Nano-Structured Lipid Carriers

Same as solid lipid nanoparticles (SLNs), nano-structured lipid carriers (NLCs) are another form of lipid-based nanoparticles. Nano-structured lipid carriers (NLC) are modified solid lipid nanoparticles consisting of a blend of solid and liquid lipids and offer an improved stability and loading capacity.
Nano-structured lipid carriers can be prepared via ultrasonic emulsion methdod.

Nano-Sized Crystals

Ultrasonic crystallization and precipitation is a highly potent way to encapsulate substances with a poor water-solubility into a coated crystal. Zheng et al. (2020) report the ultrasonic encapsulation of curcumin, a bioactive compound with many health benefits, but poor bioavailability due to low water-solubility. The research team developed a polyelectrolyte layer-by-layer (LbL) nanoshell formation to encapsulate the curcumin molecules. They state that “[u]nlike the commonly used emulsion methods, our ultrasonic assisted LbL encapsulation can achieve nanoparticles of much smaller size. For curcumin, we obtained crystalline nanoparticles with an average size of 80 nm, and ξ-potential of +30 mV or -50 mV, which ensured stability of these nanocolloids for months (kept in saturated drug solution). Formation of shells with two bilayers of biocompatible polyelectrolytes allowed slow drug release during ca 20 hours.”
The curcumin nucleation protocol: Curcumin powder was dissolved in a 60 % ethanol / water solution. After complete dissolution of the curcumin, aqueous polycations, poly(allylamine hydrochloride), PAH, or biodegradable protomine sulfate, (PS) were added . Then, the solution was sonicated with an UIP1000, a 1kW powerful ulötrasonicator from Hielscher Ultrasonic, at 100watts per mL of solution. During ultrasonication, water was slowly added to the solution. Due to the added water, the solvent becomes more polar, which reduces the solubility of curcumin. When the equilibrium concentration exceeds the solubility threshold ra supersaturation of curcumin is obtained and crystal nucleation starts. Under high power ultrasonication, the drug particle growth is stopped at initial stages.
Read more about the ultrasonic precipitation and crystallisation of nano-crystals!

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