a technique to generate a novel delivery platform for a delayed burst release of molecules

A Technique to Generate a Novel Delivery Platform for a Delayed Burst Release of Molecules

To fabricate the polybubbles, first, use a 1-milliliter transfer pipette to add 800 microliters of 10% CMC into a 0.92-milliliter glass vial. To synthesize PCLTA, mix 1,000 grams per milliliter of 14 kilodalton PCL in 200 microliters of DCM. To synthesize PLGADA, mix 1,000 grams per milliliter of 5 kilodalton PLGADA into 200 microliters of chloroform.
Mix photoinitiator with the polymer mixture of interest at a 0.005-to-1 ratio and load 200 microliters of the resulting solution into a 1-milliliter glass syringe mounted on a syringe pump connected to a dispensing stainless steel tube with an inner diameter of 0.016-inch.
Using a micro-motor to control the forward and backward motion of the polymer tube, inject the polymer into the 10% CMC in the glass vial to form the polybubbles and cure the polybubbles under ultraviolet light at a 254-nanometer wavelength for 60 seconds at 2 watts per square centimeter. Then, flash-freeze the polybubbles in liquid nitrogen for 30 seconds and lyophilize the carriers overnight at 0.01 millibar vacuum and minus 85 degrees Celsius.
For centering of the cargo of interest within a polybubble, mix the cargo with 5% CMC and a rotator overnight to increase the viscosity of the cargo before manually injecting 2 microliters of the cargo mixture into the poly bubble. When all of the cargo has been injected, recure, flash-freeze, and lyophilize the injected polybubble as just demonstrated.
The next morning, use forceps to separate the polybubble from the dried CMC and wash the polybubble with deionized water to remove any residual CMC. Then, cut the polybubble in half and image the halves by confocal microscopy to ensure that the cargo is centered.
For small-molecule cargo release, incubate the polybubbles with centered acriflavine and 400 microliters of PBS at 37 degrees Celsius for the appropriate incubation length. At each experimental time point, collect the supernatant, and add 400 microliters of fresh PBS to the vial. Then, use a plate reader to quantify the fluorescence intensity of the collected supernatant.

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Universidad de Cartagena UDEC

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Immunogenics and Vaccine Development

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1. Polycaprolactone triacrylate (PCLTA) synthesis
<ol>
	<li>Dry 3.2 mL of 400 Da polycaprolactone (PCL) triol overnight at 50 &#176;C in an open 200 mL round bottom flask and potassium carbonate (K2CO3) in a glass vial at 90 &#176;C.</li>
	<li>Mix the triol with 6.4 mL of dichloromethane (DCM) and 4.246 g of K2CO3 under argon.</li>
	<li>Mix 2.72 mL of acryloyl chloride in 27.2 mL of DCM and add dropwise to the reaction mixture in the flask over 5 min.</li>
	<li>Cover the reaction mixture with aluminum foil and leave it undisturbed at room temperature for 24 h under argon.</li>
	<li>After 24 h, filter the reaction mixture using a filter paper on a Buchner funnel under vacuum to discard excess reagents.</li>
	<li>Precipitate filtrate from step 1.5 that contains the endcapped polymer in diethyl ether in a 1:3 (vol/vol) and rotavap at 30 &#176;C to remove the diethyl ether.</li>
</ol>
2. Formation of the polybubble
NOTE: Injecting polymer in the deionized (DI) water would cause the polybubbles to migrate to the bottom of the vial resulting in flattened bottom. Use 10% (wt/vol) carboxymethyl cellulose (CMC) to fill the glass vial instead to avoid polybubble flattening.
<ol>
	<li>Prepare 10% (wt/vol) CMC solution in DI water.</li>
	<li>Fill a 0.92 mL glass vial with 0.8 mL of 10% CMC using a 1 mL transfer pipet.</li>
	<li>Mix 1000 mg/mL of 14 kDa PCL in DCM and synthesize PCLTA in a 1:3 (vol/vol) for a total volume of 200 &#181;L or prepare 200 &#181;L of 1000 mg/mL of 5 kDa poly (lactic-co-glycolic acid) diacrylate (PLGADA) in chloroform.</li>
	<li>Mix the 2-hydroxy-4&#8242;-(2-hydroxyethoxy)-2-methylpropiophenone (photoinitiator) with the polymer (PLGADA or PCL/PCLTA) mixture in 0.005:1 (vol/vol).</li>
	<li>Load 200 &#181;L of polymer mixture into a 1 mL glass syringe mounted on a syringe pump that is connected to a dispensing stainless-steel tube with an inner diameter of 0.016 inch.</li>
	<li>Use a micromotor to control the forward and backward motion of the polymer tube to inject polymer into the 10% CMC in the glass vial to form the polybubble.</li>
	<li>Cure the polybubbles under ultraviolet (UV) at 254 nm wavelength for 60 s at 2 W/cm2.</li>
	<li>Flash freeze the polybubbles in liquid nitrogen and lyophilize overnight at 0.010 mBar vacuum and at -85 &#176;C.</li>
	<li>Separate the polybubbles from the dried CMC using forceps and wash the polybubbles with DI water to remove any residual CMC. Note that other polymers can be used likely with modifications to alter the release kinetics.</li>
</ol>
3. Modulation of polybubble diameter
<ol>
	<li>Fill a 0.92 mL glass vial with 10% CMC using a 1 mL transfer pipet.</li>
	<li>Mix PCL/PCLTA in a 1:3 (vol/vol) with 1000 mg/mL 14kDa PCL and synthesize PCLTA. Mix the photoinitiator with a polymer mixture in a 0.005:1 (vol/vol).</li>
	<li>Load the polymer mixture into a 1 mL glass syringe mounted on a syringe pump that is connected to a dispensing stainless-steel tube with an inner diameter of 0.016 inch.</li>
	<li>Use a micromotor to control the forward and backward motion of the polymer tube to inject polymer into the 10% CMC in the glass vial to form the polybubble.</li>
	<li>To obtain polybubbles with various diameters, vary dispensing rate from 0.0005 to 1 &#181;L/s.</li>
	<li>Take images of the vial with the polybubbles with varying diameter.</li>
	<li>Use ImageJ to quantify the diameter of the polybubbles and use the size of the vial as scale.</li>
</ol>
4. Centering cargo within polybubble
<ol>
	<li>Modulation of PCL/PCLTA viscosity using K2CO3:&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
	NOTE: Viscosity of PLGADA does not have to be modified using K2CO3&#160;because the viscosity of 5 kDa PLAGDA at 1000 mg/mL is sufficient for centering the cargo.
	<ol>
		<li>Add K2CO3&#160;(that was isolated after the PCLTA reaction) to the PCLTA at varying concentrations including 0 mg/mL, 10 mg/mL, 20 mg/mL, 40 mg/mL, and 60 mg/mL.</li>
		<li>Measure the dynamic viscosities of the solutions by changing the shear rate from 0 to 1000 1/s using rheometry.</li>
		<li>Manually inject the cargo in the middle (refer to step 4.2 to prepare the cargo mixture) of the polybubbles that were formed using the PCL/PCLTA solutions with different concentrations of K2CO3&#160;(step 4.1.1). Determine the optimal concentration of K2CO3&#160;by observing which solution from step 4.1.1 can result in retention of the cargo in the middle.</li>
	</ol>
	</li>
	<li>Centering of the cargo (already shown feasibility with small molecules) with CMC
	<ol>
		<li>Mix the cargo with 5% (wt/vol) CMC in a rotator overnight to increase the viscosity of the cargo.</li>
		<li>Manually inject 2 &#181;L of cargo mixture in the polybubble and proceed with UV curing at 254 nm wavelength for 60 s at 2 W/cm2.</li>
		<li>Flash freeze the polybubbles in liquid nitrogen for 30 s and lyophilize overnight at 0.010 mBar vacuum and at -85 &#176;C.</li>
		<li>Separate the polybubbles from the dried CMC using forceps and wash with DI water to remove any residual CMC.</li>
		<li>Cut the polybubble in half and image the halves using confocal microscopy to ensure that the cargo is centered (refer to step 6 for excitation and emission wavelengths used).</li>
	</ol>
	</li>
</ol>
5. Cargo Formulation
NOTE: Polybubble formulation can house various cargo types, including small molecules, proteins, and nucleic acids.
<ol>
	<li>Based on previous studies, in the case of protein cargo, use excipients including polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and glycopolymers&#160;to improve the stability of protein during polybubble formulation.</li>
	<li>Form polybubbles based on the protocol in step 2.</li>
	<li>Prepare the antigen solution by adding 17.11 g of trehalose to 625 &#181;L of HIV gp120/41 antigen.</li>
	<li>Manually inject 1 &#181;L of antigen solution in the middle of the polybubble.</li>
	<li>Open polybubbles on days 0, 7, 14, and 21, and record the fluorescence of antigen with excitation and emission wavelengths 497 nm and 520 nm, respectively.</li>
	<li>Determine the functionality of the antigen using enzyme-linked immunosorbent assay (ELISA) and use 5% nonfat milk as a blocking buffer.</li>
</ol>
6. Release of cargo
NOTE: Small molecule or antigen can be used as the cargo type
<ol>
	<li>Small molecule
	<ol>
		<li>Incubate polybubbles with centered acriflavine in 400 &#181;L of phosphate buffer saline (PBS) at 37 &#176;C, 50 &#176;C for PLGADA polybubbles and at 37 &#176;C, 50 &#176;C, 70 &#176;C for PCL/PCLTA polybubbles.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 
		NOTE: The reason why we recommend testing above body temperatures is to a) simulate the temperature (50 &#176;C) at which the polybubble reaches while lasering the gold nanorods (AuNRs) within PCL and PLGA; and b) accelerate the degradation process of PCL (50 &#176;C, 70 &#176;C).</li>
		<li>At each time point, collect the supernatants and replace with 400 &#181;L of fresh PBS.</li>
		<li>Use a plate reader to quantify the fluorescence intensities in the collected supernatants. 
		NOTE: Use ex/em of 416 nm/514 nm for acriflavine.</li>
	</ol>
	</li>
	<li>Antigen
	<ol>
		<li>Incubate polybubbles with centered bovine albumin serum (BSA)-488 in 400 &#181;L of PBS at 37 &#176;C, 50 &#176;C for PLGADA polybubbles and at 37 &#176;C, 50 &#176;C for PCL/PCLTA polybubbles.</li>
		<li>At each time point, collect the supernatants and replace with 400 &#181;L fresh PBS.</li>
		<li>Use a plate reader to quantify the fluorescence intensities in the collected supernatants. Use ex/em of 497 nm/520 nm for BSA-488. 
		NOTE: Release study at 70 &#176;C for PCL/PCLTA polybubbles should not be conducted to avoid exposing the antigen to extreme temperature.</li>
	</ol>
	</li>
</ol>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Step Ultra Tetramethylbezidine (TMB)-Enzyme-Linked Immunosorbent Assay (ELISA) Substrate Solution</td>
			<td>Thermo scientific</td>
			<td>34028</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Hydroxy-2-methylpropiophenone</td>
			<td>TCI AMERICA</td>
			<td>H0991</td>
			<td></td>
		</tr>
		<tr>
			<td>450 nm Stop Solution for TMB Substrate</td>
			<td>Abcam</td>
			<td>ab17152</td>
			<td></td>
		</tr>
		<tr>
			<td>Acryloyl chloride</td>
			<td>Sigma Aldrich</td>
			<td>A24109-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Acriflavine</td>
			<td>Chem-Impex International</td>
			<td>22916</td>
			<td></td>
		</tr>
		<tr>
			<td>Anhydrous ethyl ether</td>
			<td>Fisher Chemical</td>
			<td>E138-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HIV1 gp120 antibody conjugated to horseradish peroxidase (HRP)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Fisher BioReagents</td>
			<td>BP9700100</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA-CF488 dye conjugates</td>
			<td>Invitrogen</td>
			<td>A13100</td>
			<td></td>
		</tr>
		<tr>
			<td>Carboxymethylcellulose (CMC)</td>
			<td>Millipore Sigma</td>
			<td>80502-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Chemical</td>
			<td>C2984</td>
			<td></td>
		</tr>
		<tr>
			<td>Coating buffer</td>
			<td>Abcam</td>
			<td>ab210899</td>
			<td></td>
		</tr>
		<tr>
			<td>Dichloromethane (DCM)</td>
			<td>Sigma Aldrich</td>
			<td>270997-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Diethyl ether</td>
			<td>Fisher Chemical</td>
			<td>E1384</td>
			<td></td>
		</tr>
		<tr>
			<td>Legato 100 Syringe Pump</td>
			<td>KD Scientific</td>
			<td>14 831 212</td>
			<td></td>
		</tr>
		<tr>
			<td>Nonfat dry milk</td>
			<td>Andwin Scientific</td>
			<td>NC9022655</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium carbonate</td>
			<td>Acros Organics</td>
			<td>AC424081000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate saline buffer</td>
			<td>Fisher BioReagents</td>
			<td>BP3991</td>
			<td></td>
		</tr>
		<tr>
			<td>(Poly(caprolactone)</td>
			<td>Sigma Aldrich</td>
			<td>440744-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>(Poly(caprolactone) triol</td>
			<td>Acros Organics</td>
			<td>AC190730250</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly (lactic-co-glycolic acid) diacrylate</td>
			<td>CMTec</td>
			<td>280050</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant HIV1 gp120 + gp41 protein</td>
			<td>Abcam</td>
			<td>ab49054</td>
			<td></td>
		</tr>
		<tr>
			<td>Trehalose</td>
			<td>Acros Organics</td>
			<td>NC9022655</td>
			<td></td>
		</tr>
	</tbody>
</table>

Source: Arun Kumar, S. et al., <a href="https://appjove.unicartagenaproxy.elogim.com/v/60569">Production of Near-Infrared Sensitive, Core-Shell Vaccine Delivery Platform.</a>&#160;J. Vis. Exp. (2020)
This video demonstrates a method for the generation of a polymer bubble or polybubble-based novel platform that enables delayed burst release. Polyester-based polymers are used to form the polybubbles with core-shell structure, and small molecules and antigens can be used as cargo.

1. Polycaprolactone triacrylate (PCLTA) synthesis

	Dry 3.2 mL of 400 Da polycaprolactone (PCL) triol overnight at 50 &#176;C in an open 200 mL round ...

Attach a syringe with a dispensing tube to a pump. The syringe contains a photoinitiator-polyester blend, a unique polymer mix.
Dispense the blend into a viscous aqueous solution of a cellulose-based polymer. This induces phase separation, forming a polymeric spherical structure called a polybubble.
Ultraviolet irradiation activates the photoinitiator, triggering the polymer to crosslink and form a shell surrounding the inner core.
Next, insert a needle to inject a viscous solution of fluorophore-tagged molecules into the polybubble core.
Irradiate again to restore the polybubble integrity. Freeze-dry to remove excess water and recover the polybubble.
To check the release profile, incubate the polybubble in a buffer.
Upon contact with an aqueous phase, the polymer shell undergoes erosion, thereby forming pores. Over time, these pores interconnect,&#160; releasing the labeled molecules.
Regularly collect the supernatant and quantify the fluorescence intensities.
A sudden increase in fluorescence following extended incubation confirms the delayed burst release.

29 Views. تقنية لإنشاء منصة توصيل جديدة لإطلاق الانفجار المتأخر للجزيئات

Video: تقنية لإنشاء منصة توصيل جديدة لإطلاق الانفجار المتأخر للجزيئات - Experiment

Formulating and Characterizing Lipid Nanoparticles for Gene Delivery using a Microfluidic Mixing Platform

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high encapsulation efficiency. Use of lipid nanoparticles (LNPs) to encapsulate nucleic acids is advantageous to protect the RNA or DNA from degradation, while also promoting cellular uptake. LNPs often contain multiple lipid components including an ionizable lipid, helper lipid, cholesterol, and polyethylene glycol (PEG) conjugated lipid. LNPs can readily encapsulate nucleic acids due to the ionizable lipid presence, which at low pH is cationic and allows for complexation with negatively charged RNA or DNA. Here LNPs are formed by encapsulating messenger RNA (mRNA) or plasmid DNA (pDNA) using rapid mixing of the lipid components in an organic phase and the nucleic acid component in an aqueous phase. This mixing is performed using a precise microfluidic mixing platform, allowing for nanoparticle self-assembly while maintaining laminar flow. The hydrodynamic size and polydispersity are measured using dynamic light scattering (DLS). The effective surface charge on the LNP is determined by measuring the zeta potential. The encapsulation efficiency is characterized using a fluorescent dye to quantify entrapped nucleic acid. Representative results demonstrate the reproducibility of this method and the influence that different formulation and process parameters have on the developed LNPs.

Lipid nanoparticles are developed using a microfluidic mixing platform approach for mRNA and DNA encapsulation.

صياغة وتميز الجسيمات النانوية الدهنية لتسليم الجينات باستخدام منصة خلط Microfluidic

Lipid-based drug carriers have been used for clinically and commercially available delivery systems due to their small size, biocompatibility, and high ...

JoVE Journal

Bioengineering

Delivery of Antibodies into the Murine Brain via Convection-enhanced Delivery

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an approach provides a safe delivery method by-passing the blood brain barrier (BBB), thus allowing treatment with therapeutics with poor BBB-permeability or those for which systemic exposure is not desired, e.g., due to toxicity. CED requires optimization of the catheter design, injection protocol, and properties of the infusate. With this protocol we describe how to perform CED of a solution containing up to 20 &#181;g of an antibody into the caudate putamen of mice. It describes preparation of step catheters, testing them in vitro and performing the CED in mice using a ramping injection program. The protocol can be readily adjusted for other infusion volumes and can be used for injecting various tracers or pharmacologically active or inactive substances, including chemotherapeutics, cytokines, viral particles, and liposomes.

Convection-enhanced delivery (CED) is a method enabling effective delivery of therapeutics into the brain by direct perfusion of large tissue volumes. The procedure requires the use of catheters and an optimized injection procedure. This protocol describes a methodology for CED of an antibody into a mouse brain.

تسليم الأجسام المضادة إلى الدماغ مورين عن طريق الحمل الحراري تعزيز التسليم

Convection-enhanced delivery (CED) is a neurosurgical technique enabling effective perfusion of large brain volumes using a catheter system. Such an ...

Neuroscience

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated with antigens. Ophiopogonin D (OP-D), a purified component extracted from the plant Ophiopogon japonicus, has been found to be useful as a vaccine adjuvant. The problems of the low solubility and toxicity of OP-D can be effectively overcome by using a low-energy emulsification method to prepare nanoemulsion ophiopogonin D (NOD). In this article, a series of in vitro protocols for cellular activity evaluation are examined. The cytotoxic effects of L929 were determined using a cell counting kit-8 assay. Then, the secreted cytokine levels and corresponding immune cell numbers after the stimulation and culture of splenocytes from immunized mice were detected by ELISA and ELISpot methods. In addition, the antigen uptake ability in bone marrow-derived dendritic cells (BMDCs), which were isolated from C57BL/6 mice and matured after incubation with GM-CSF plus IL-4, was observed by laser scanning confocal microscopy (CLSM). Importantly, macrophage activation was confirmed by measuring the levels of IL-1&#946;, IL-6, and tumor necrosis factor alpha (TNF-&#945;) cytokines by ELISA kits after coculturing peritoneal macrophages (PMs) from blank mice with the adjuvant for 24 h. It is hoped that this protocol will provide other researchers with direct and effective experimental approaches to evaluate the cellular response efficacies of novel vaccine adjuvants.

In Vitro Cellular Activity Evaluation of the Nanoemulsion Vaccine Adjuvant Ophiopogonin D

The protocol presents detailed methods for evaluating whether the nanoemulsion ophiopogonin D adjuvant promotes effective cellular immune responses.

في المختبر تقييم النشاط الخلوي لمساعد لقاح المستحلب النانوي Ophiopogonin D

As a principal ingredient of vaccines, adjuvants can directly induce or enhance the powerful, widespread, innate, and adaptive immune responses associated ...

A Technique to Generate a Novel Delivery Platform for a Delayed Burst Release of Molecules

Transcript

A Technique to Generate a Novel Delivery Platform for a Delayed Burst Release of Molecules