Synthesis and Characterization of Polymer Nanocarriers for the Targeted Delivery of Therapeutic Enzymes
6. Add PEG sample from Step 1 to wells in appropriate vol- vol-umes such that approximately 10–0.1 g of PEG is present
3.6. Characterization of Targeted PNC In
Vitro – PNC Binding to Cells
3.6.1. Fluorescence Detection of PNC Binding to Cells
To visualize binding and to show its specificity we used epifluo-rescence imaging.
1. Plate REN and REN-hPECAM cells on 24-well dishes with inserted 12-mm glass coverslips and cultivate to confluence.
2. Add anti-PECAM/PNC to confluent cells and incubate for 1 h in regular cell medium at 37◦C.
3. Wash out unbound nanocarriers 5 times with fresh RPMI medium, fix cells, and stain with secondary FITC-labeled antibody against mouse IgG.
4. Visualize the bound nanocarriers in REN-hPECAM cells vs.
REN cells as a negative control using fluorescence micro-scope.
3.6.2. Detection of PNC Binding to Cells by Radioisotope Tracing
Quantification of the binding to human endothelial cells was per-formed by radioactive tracing of nanocarriers.
1. Plate HUVEC on 24-well dishes and cultivate until culture is confluent.
2. Add anti-PECAM/PNC or control IgG/PNC labeled with
125I-IgG-SA tracer to cells at serial dilutions, starting with
∼25,000 cpm per well and incubate for 1 h in regular cell medium at 37◦C.
3. Wash out unbound nanocarriers 5 times with fresh RPMI medium and lyse cells with 1% Triton X-100, 1.0 N NaOH.
4. Transfer cell lysate in glass tubes and measure radioactiv-ity of bound material using Wallac 1470 WizardTM gamma counter. Bound material is expressed as number of bound particles per cell.
160 Simone et al.
4. Notes
1. Formulation. Do not allow second emulsion test tube con-taining surfactant to sit on ice longer than 15–30 min prior to the actual second emulsion. When the surfactant temper-ature of the second emulsion is at 4◦C, the addition of the primary emulsion (–80◦C) brings the entire solution below the gel point for the surfactant solution resulting in poor mixing.
2. Serial centrifugation size fractionation. Serial centrifugation to separate different size populations (28) is best used when synthesized particles possess two distinct population sizes, as is the case in this freeze–thaw solvent evaporation PNC for-mulation. Centrifugation time and speed can be determined by calculating the time needed to settle larger particles using Stokes’ law:
V = 2ga2ρ1− ρ2
9η
where V is the settling velocity, g the relative centrifugal force, a the particle radius,ρ1andρ2are the particle density and the buffer density, respectively, andη is the buffer vis-cosity. After centrifugation, the supernatant can be collected and centrifuged at a greater speed to collect the nanoparti-cle fraction. This procedure is an efficient and rapid method of both size selection and removal of residual surfactant.
An example of a centrifugation scheme is represented in Fig. 9.4. To remove any residual surfactant that may be bound to the PNC surface, resuspension of nanocarriers in buffer and an additional centrifugation purification step is recommended. As with any centrifugation fractionation technique, resuspension of PNC can be difficult. deally cen-trifugation rates will be selected such that PNC will be pelleted but not aggregated. However, if aggregation does occur, several laboratory techniques can be used to aid in resuspension. For instance, allow the sample to solvate for 5 min to 1 h with mild vortexing. Allowing the PNC to rehy-drate slowly can aid in the hydration of the PNC’s PEG shell, enhancing particle–particle repulsion. If, after this period, resuspension is still difficult, application of mild pulse sonica-tion (5 W, 1 s intervals) can be used. However, such energy addition may result in disruption of PNC, releasing cargo and/or denaturing loaded protein.
3. PNC size control. The final size of PNC is dictated by a balance of both kinetic and thermodynamic forces.
Targeted Polymer Nanocarriers for Therapeutic Enzyme Delivery 161
Centrifugation
g (x 1000)
0 5 10 15 20 25
Diameter (µm)
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
1000 g 10 min
Nano size domain (higher g or longer time)
Fig. 9.4. Centrifugation and size fractionation. The scheme represents approximate trifugation rates (g) necessary to sediment different size populations in a 10 min cen-trifugation of PLA-based particles. Higher rate, g, or longer spin time, can bring down smaller particles in the desired sub-500 nm range.
Mechanical disruption of the two-phase liquid system results in droplet formation. As more energy is added (either through faster shear rates, longer times, larger impeller diameters) the droplets are disrupted and form smaller droplets. However, as the interface between the two phases increases, the destabilizing energy also increases. Unless there is a surfactant that can function to stabilize these two phases, droplets will start to coalesce and form larger par-ticles. Yet this coalescence is rate limiting and determined by factors including aqueous phase viscosity, polymer phase viscosity, surfactant concentration, and temperature. Figure 9.5demonstrates this balance between energy and size.
To summarize these effects, smaller PNC can be obtained by higher energy rates and increased surfactant content (either the amphiphilic PNC polymer or added solution surfactant). However, as PNC size decreases, so does the internal volume to surface area ratio. With smaller inter-nal volumes, the likelihood of successfully encapsulating protein drops off significantly. Furthermore, longer dura-tion and higher shear rate homogenizadura-tions can denature enzymes, dramatically decreasing recovered activity. As such, ultimate carrier size is selected by a compromise between cel-lular/vascular compatibility and protein loading efficiency.
4. Particle yield and mass determination. Double emulsion PNC synthesis typically results in a bimodal distribution, with a significant fraction of undesired large (>1 m) par-ticles. During process optimization, reduction in this loss fraction will be a significant technological hurdle. However, proper surfactant concentration may provide one avenue of achieving higher PNC yields. Increasing PVA surfactant con-centration from 0.1 to 4 wt% increased the PNC fractional
162 Simone et al.
Rate2 time (krpm2*min)
0 200 400 600 800 1000 1200
Particle Size (nm) (Filled Circle) 300 400 500 600 700 800 900
Particle Size (nm) (Open Circle)
260 280 300 320 340
Fig. 9.5. PNC size decreases with increasing energy input. Mean diameter (mea-sured by DLS) is a function of both energy input (which scales with homogenization rate2t ) and polymer surfactant capacity (• 5% and ◦ 11% PEG content). When surfac-tant capacity of system is saturated, further increases in energy input do not decrease PNC size. However, greater PEG content (greater intrinsic surfactant capacity of PNC polymer) results in formulation of even smaller PNC carriers.
yield, but also simultaneously increased the carrier diame-ter as well. This result is most likely due to two contribut-ing factors. With greater PNC mass, the overall surface area between phases is increasing, resulting in a saturation of PVA’s surfactant capacity at larger sizes. Also, the increased viscosity of the aqueous phase reduced the rate of droplet breakup under the mixing conditions, further adding to the larger carrier size.
Polymer concentration analysis provides the only reason-able method to determine PNC yield. Yet, the PEG assay approach possesses several potential complications. Poly-meric surfactant used in PNC synthesis can participate in the barium iodide complex, resulting in overestimating the actual PNC concentration. This is of particular concern with Pluronic R, a PEG-containing surfactant. As such, care-ful particle purification in the form of extra centrifugation steps and/or dialysis may be necessary to determine the actual PNC concentration. PEG assays of the supernatants and dialysates provide an indicator of amount of surfactant released through each step. Once no measurable PEG is found, PNC can be assumed to be clear of surfactant. If dialyzing, keep in mind that Pluronic R, although less than 9 kDa, is a linear polymer and hence has a hydrodynamic radius on the order of a 500 kDa protein.
Erratic results with the PEG assay may also be obtained in the presence of high protein content (above 3 mg/mL) (31). As an alternative, an enzymatic assay may be utilized
Targeted Polymer Nanocarriers for Therapeutic Enzyme Delivery 163
for the detection of lactic acid, the monomeric building block of PLA (see Note 5).
5. Enzymatic lactic acid assay. The working buffer used in this concentration assay is very sensitive to pH, and therefore 100 mM PBS is recommended for solutions and dilutions.
1. Rapidly hydrolyze a 50 L aliquot of PNC sample by adding 200 L of 5 N NaOH and reacting overnight at 80◦C, before neutralizing with 5 N HCl. This assay is sensitive only to the L-lactic acid enantiomer, and hence this step is necessary to convert allL-lactic acid in monomeric form.
2. Prepare a 0.01 mg/mL calibration solution of L-lactic acid in DI water.
3. In a 96-well microplate, prepare lactic acid calibra-tion wells as described above in the PEG assay (Section 3.3.4).
4. Add sample from step 1 to wells in appropriate volumes such thatL-lactic acid is within the calibration range.
5. Dilute sample and calibration volumes to 50 L using 100 mM PBS.
6. Prepare working buffer of 4.75 mL of 100 mM PBS, 50L of 10 mM Amplex red in DMSO, 100 L of 10 U/mL HRP, and 100L of 50 U/mL lactate oxidase.
7. Add 50L of working buffer to each well.
8. Allow to develop for 10 min and measure absorbance at 530 nm on the microplate reader. A 550 nm filter on the reader is adequate.As mentioned above, this method is very pH sensitive, and 100 mM PBS may not be ade-quate. In such an event, careful titration of sample to neu-tral pH, after saponification, will be necessary. Also, the reagents of the working buffer, primarily lactate oxidase, are extremely sensitive to denaturation at room tempera-ture and hence should not be prepared until ready to use.
Make sure working buffer enzymes are stored at –20◦C.
5. Conclusions
Several factors must be weighed when formulating the “optimal”
PNC. Properties such as yield, enzyme loading, and PNC siz-ing can be tuned through process manipulation. Yet these pro-cess manipulations (e.g., energy input, surfactant concentration) can have complex and competing effects in terms of sizing and enzyme activity. Along with the notion that the definition of
164 Simone et al.
optimal is highly dependent upon the therapy being pursued, it is not possible to know a priori what the ideal compromise in PNC properties is. Coupled with the capacity to vary biodistribution through the non-aggregating coupling of antibodies to the carrier surface, in vivo studies must always be performed as a feedback mechanism to tune the synthesis of carrier for each specific thera-peutic goal. Only through this continual interaction between the engineering and the biology can PNC delivery strategies result in clinically relevant solutions.
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