Combination of Roll Grinding and High-Pressure Homogenization Can Prepare Stable Bicelles for Drug Delivery
, Shin-ichiro Kimura 1, Shigeru Itai 1, Hiromu Kondo 1 and Yasunori Iwao 1,*Round 1
Reviewer 1 Report
The manuscript describes the physico-chemical characterization of various bicelles containing nifedipine made up of different lipid mixture and obtained by roll grinding and high-pressure homogenization. The rationale was provided and the experiments supported the thesis provided by the authors. The following criticisms should be properly addressed in order to improve the quality of the manuscript.
Page 3, line 117 – “The NI-LN suspension was filtered with a 0.2-μm polytetrafluoroethylene or cellulose acetate membrane filter….” – More details are required (equipment, for example syringe or extruder, cycles, etc). Moreover, the authors did not describe any purification methods (centrifugation, dialysis, etc). Have been the non-encapsulated drugs separated by the systems? It could be detrimental for the EE% analysis.
Page 3, line 120 – “2.3. Freeze-drying and reconstitution of NI-lipid nanoparticles” The authors should justify the choice of sucrose instead of other cryoprotectants (i.e. mannose, glucose, trehalose, etc) and the concentration used.
Page 3, line 126 – “2.4. Measurement of mean particle size and zeta potential” – the dilution used should be added.
Page 6, line 214 – “On the other hand, the data by DLS shows the value of total particles including bicelles and micelles and was 47.5 nm”. The authors should evaluate the data as number(%), volume(%) and intensity(%) in order to furnish a plausible size distribution of the various systems.
The modulation of the physico-chemical parameters of the various formulations should be evaluated in saline (0.9% NaCl).
Tables 5 and 6 – the type of ratio (molar) should be reported.
The quality of Figure 8 panel B must be improved.
Author Response
Response to Reviewers' Comments
We thank the reviewers for the very valuable comments. We have addressed all the issues raised by the reviewers in a point-by-point manner as noted below. The changes made in the manuscript have been marked in red.
Reviewer #1. We thank the reviewer for his or her useful comments.
Response:
Comments | Our response | |
1 | Page 3, line 117 – “The NI-LN suspension was filtered with a 0.2-μm polytetrafluoroethylene or cellulose acetate membrane filter….” – More details are required (equipment, for example syringe or extruder, cycles, etc). Moreover, the authors did not describe any purification methods (centrifugation, dialysis, etc). Have been the non-encapsulated drugs separated by the systems? It could be detrimental for the EE% analysis. | We are sorry for insufficient explanation. After high-pressure homogenization with a pass cycle of 100, the filtration was carried out by suction aspirator using a 0.2-µm cellulose acetate membrane filter (Toyo Roshi Kaisha Ltd.) to remove the particles whose size was over 200 nm. Based on the reviewer’s comment, the following sentence has been revised in the revised manuscript. (page 3, line 119) : The NI-LN suspension was filtered by suction aspirator with a 0.2-µm polytetrafluoroethylene or cellulose acetate membrane filter (Toyo Roshi Kaisha Ltd.) to remove the particles whose size was over 200 nm. In addition, after filtration, we did not perform any purification methods like centrifugation and dialysis. It means that the sample solution contains NI-encapsulated LNs which can be pass through the 0.2-μm filter and non-encapsulated drugs. However, when measuring the Entrapment efficiency (EE)(%) after ultrafiltration by ultrafilter, EE values were over 97% in Table 2 (original manuscript) and free drug percentage (%) was just 2-3%. Therefore, since the free drug percentage was quite low, we think additional purification methods are not necessary in this study.Thank you for your comments. |
2 | Page 3, line 120 – “2.3. Freeze-drying and reconstitution of NI-lipid nanoparticles” The authors should justify the choice of sucrose instead of other cryoprotectants (i.e. mannose, glucose, trehalose, etc) and the concentration used. | Thank you for your comments. Actually, we previously reported that when adding some types of cryoprotectants (glucose, fructose, maltose or sucrose) at various concentrations to NI-LNs and then freeze-drying, good re-dispersibility of NI-LNs with disaccharides (maltose and sucrose) were shown compared with monosaccharides (glucose and sucrose)(Ohshima H, et al. (2009), Ref #28 in original manuscript). In addition, Barman et al also reported that in comparison to monosaccharide, disaccharide showed higher cryoprotectant activity when adding at a concentration of 5% (w/v). Therefore, we used sucrose as one of good cryoprotectants in this study. Based on the reviewer’s comment, the following sentence has been revised and new reference has been added as #37 in the revised manuscript. (Page 3, line 121) : Freeze-drying was performed with 2 ml of the NI-LN suspensions placed in a vial containing 100 mg of sucrose (5%, w/v) as a cryoprotectant. Sucrose was selected based on the previous reports [28][37]. (Reference) 37. Barman, R.; Iwao, Y.; Funakoshi, Y.; Ranneh, A.; Noguchi, S.; Wahed, M.; Itai, S. Development of Highly Stable Nifedipine Solid–Lipid Nanoparticles. Chem. Pharm. Bull. 2011, 47, 4691–4693. |
3 | Page 3, line 126 – “2.4. Measurement of mean particle size and zeta potential” – the dilution used should be added. | When measuring mean particle size, PDI and zeta potential by zetasizer, the values of mean count rate of intact samples were 200–500 kcps which are suitable for this measurement. Therefore, we did not perform the dilution in this study. Based on the reviewer’s comment, the following sentence has been added in the revised manuscript. (Page 3, line 133): The mean particle size and the zeta potential of LN suspensions were measured with dynamic light scattering (DLS) (Zetasizer nano ZS, Malvern Instruments Ltd., Worcestershire, UK) at a scattering angle of 90° at room temperature (25°C). Mean particle sizes were based on the scattering intensity, while the zeta potential was based on electrophoretic mobility. Since the values of mean count rate of the samples were 200–500 kcps which are suitable for this measurement, intact samples without dilution were used in this measurement. We repeated measurement three times per a sample. |
4 | Page 6, line 214 – “On the other hand, the data by DLS shows the value of total particles including bicelles and micelles and was 47.5 nm”. The authors should evaluate the data as number (%), volume (%) and intensity (%) in order to furnish a plausible size distribution of the various systems. | Thank you for your comment. We agree with your idea and we always have to consider which analyses (intensity-based, Volume-based and Number-based) by zetasizer would be appropriate for measuring nanoparticles. The values of mean particle size and PDI were determined by scattering intensity, as described in line 129 of original manuscript. Definitely, since our LNs has some types of particles, like bicelles, micelles and liposomes, number-based analyses would be appropriate if we show the data of the particle size distribution. However, in this study, we want to just determine the mean particle size of LNs, and even if the intensity-based analysis was changed to volume-based and number-based analyses, all data of mean particle sizes (z-values) of NI-LNs were same because cumulant method was applied in all analyses. In addition, all the data of quality report were “good”, meaning this analysis was acceptable. Taken together, in this study, we want to show only the data of mean particle size and we think intensity-based analysis is enough. We hope the reviewer understanding our thought.
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5 | The modulation of the physico-chemical parameters of the various formulations should be evaluated in saline (0.9% NaCl). | Thank you for your comments. As you suggested, as additional experiments, we measured the particle size of NI-LNs dispersed in PBS. The mean particle size was 59.4 nm and almost same as the result of NI-LN in distilled water. However, as you pointed out, we agree with your idea that dispersion medium affects the physicochemical properties of NI-LNs. Actually, as new experiments, we have been evaluating the physicochemical properties of LNs in simulated gastrointestinal fluids (Fasted State Simulated Intestinal Fluid (FaSSIF) or Fed State Simulated Intestinal Fluid (FeSSIF)), and we want to report them in the near future as a further study. We believe you understand our thought. |
6 | Tables 5 and 6 – the type of ratio (molar) should be reported. | Thank you. The word “HSPC/DPPG (molar ratio)” has been added in revised Table 5 and 6 in the revised manuscript. |
7 | The quality of Figure 8 panel B must be improved. | We agree with your idea. However, it would be difficult to additionally take cryo-TEM images and replace new one for the following reasons. One is revision due of this manuscript is within only one week. Because this cryo-TEM machine is not mine and common use apparatus, we cannot use this machine in this period. In addition, as another reason, even if we can use this machine and perform additional experiments, as you know, it would spend a huge amount of time for taking the images by cryo-TEM, because it is technically difficult to observe such kind of small enough particle. In this image (Fig. 8B), to easily find and focus on these small particles, the magnification was a little bit decreased and to justify the length of scale bar of other images, the image was expanded, which might become blurred. However, we think this image was still enough to show the morphology of these small particles. So, we hope reviewer understand our situation and thought. |
Author Response File: 
Author Response.pdf
Reviewer 2 Report
In the manuscript “Combination of roll grinding and high-pressure homogenization can prepare stable bicelles for drug delivery” the authors reported the synthesis and characterization of bicelles for nifedipine and phenytoin delivery.
Although the paper is well written, I think that the authors confused the several concepts, such as nanoparticles, bicelles and liposomes. In my opinion, the manuscript is very unclear.
Nanoparticles are solid materials in nanoscale and bicelles are amphipathic structures composed of two phospholipids having a critical micelle concentration to be formed. The presence of the phospholipid confers high fluidity of the lipids. Thus, the two concepts (nanoparticles and bicelles) are not the same thing. I suggest that the authors replace “lipid nanoparticles” for “lipid nanostructures” or “lipid aggregates”
Another negative point is that the authors indicate that the authors said in Abstract that “The NI-LNs exhibited high entrapment efficiency, long-term stability, and enhanced NI bioavailability”. In the present paper, the authors did not present any in vivo studies for bioavailability. Therefore, I suggest that the author remove this affirmation from the manuscript.
My main commentaries are given below:
Abstract
The abstract is very confusing. In the beginning of Abstract, the author said “To improve the solubility of the drug nifedipine (NI), NI-encapsulated lipid nanoparticles (NI-LNs) were prepared from neutral hydrogenated” However, in the final of the Abstract, the authors said about other dug: phenytoin. Please, reformulate it.
The authors said that the NI-LNs enhanced the bioavailability. I think this is not true. The authors did not show any data for bioavailability studies. Please, remove it.
Introduction
(Line 69-73) I think that the reference (27) is not so recent. Please, rephrase the sentence.
Material and Methods
(Line 126) Please, supply the real temperature, the angle and the concentration of the formulations that were measured in DLS. How many measurements of each sample did the authors do?
Did the authors dilute the samples before the analysis?
Results
(Line 188) Liposomes are not particles. Liposomes are vesicles in nanoscale. Please, rephrase the sentence.
(line 206) How can the authors affirm that the mean particle size became slightly bigger after freeze-drying, but the bicelle structure was preserved”? Please, clarify it.
In addition, the authors said that “Thus, freeze-drying and the addition of a lyophilization stabilizer did not affect the NI-LN structure”. How the lyophilization stabilizers did not affect the NI-LN structure if the particle size became bigger? For me it is not clear.
How did the author measure the size relating to particles, micelles and bicelles as indicated inTable 1, 2 and 3?
(Line 271) Liposomes? Please, explain it for better understanding.
Author Response
We thank the reviewers for the very valuable comments. We have addressed all the issues raised by the reviewers in a point-by-point manner as noted below. The changes made in the manuscript have been marked in red.
Reviewer #2. We thank the reviewer for his or her useful comments.
1. Although the paper is well written, I think that the authors confused the several concepts, such as nanoparticles, bicelles and liposomes. In my opinion, the manuscript is very unclear. Nanoparticles are solid materials in nanoscale and bicelles are amphipathic structures composed of two phospholipids having a critical micelle concentration to be formed. The presence of the phospholipid confers high fluidity of the lipids. Thus, the two concepts (nanoparticles and bicelles) are not the same thing. I suggest that the authors replace “lipid nanoparticles” for “lipid nanostructures” or “lipid aggregates” | Thank you for your comment. We agree with your idea. As you pointed out, definition of the lipid-based nanocarriers must be very important. Lipid-based nanocarriers are broadly comprised of liposomes, niosomes, cubosomes, nanoemulsions, nanomicelles, and lipid nanoparticles (LNs) (Gan et al., Drug Discov. Today. 2013). We think that “solid lipid nanoparticles (SLN)” is comprised of LNs and the SLN structure is composed of a solid lipid core, which may contain triglycerides, glyceride mixtures, or waxes that are solid at both room temperature and human body temperature (Wissing SA, et al., Solid lipid nanoparticles for parenteral drug delivery. Adv Drug Deliv Rev. 2004). From this perspective, although two phospholipids used in this study are solid at both room temperatures, we doubt that whether our nanocarrier is SLN. Therefore, we used the term “Lipid nanoparticles (LNs)” which means “lipid-based nanoparticles” until now (Ohshima, H. et al., Int. J. Pharm. 2009, Funakoshi, Y. et al., Int. J. Pharm. 2013,). In addition, throughout a series of experiment in this study, we found for the first time, that these lipid-based nanoparticles were defined as bicelles. Therefore, we think the term “lipid aggregates” as you suggested is also good, but we want to use the word “LNs” after revising the meaning from “lipid nanoparticle” to “lipid-based nanoparticles” in the revised manuscript as follows.
(Abstract) : To improve the solubility of the drug nifedipine (NI), NI-encapsulated lipid-based nanoparticles (NI-LNs) have been prepared
We hope reviewer understand our thought. If our thought is something wrong, please let me know again. Thank you very much for your useful comments. |
2. The abstract is very confusing. In the beginning of Abstract, the author said “To improve the solubility of the drug nifedipine (NI), NI-encapsulated lipid nanoparticles (NI-LNs) were prepared from neutral hydrogenated” However, in the final of the Abstract, the authors said about other dug: phenytoin. Please, reformulate it. The authors said that the NI-LNs enhanced the bioavailability. I think this is not true. The authors did not show any data for bioavailability studies. Please, remove it. | We are so sorry for confusing you. In the previous studies, lipid nanoparticles have already been prepared to enhance the solubility of poorly water-soluble drug. These nanoparticles have exhibited high entrapment efficiency, long-term stability, and enhanced NI bioavailability; however, it remains unclear about the structure. In this study, to better understand their structures, cryo transmission electron microscopy and atomic force microscopy were performed. So, at the beginning part of our abstract, the descriptions must be confusing. Therefore, these descriptions have been revised as follows. Thank you so much.
(Abstract) : To improve the solubility of the drug nifedipine (NI), NI-encapsulated lipid-based nanoparticles (NI-LNs) have been prepared from neutral hydrogenated soybean phosphatidylcholine and negatively charged dipalmitoylphosphadylglycerol at a molar ratio of 5/1 using by roll grinding and a high-pressure homogenization. The NI-LNs have exhibited high entrapment efficiency, long-term stability, and enhanced NI bioavailability. To better understand their structures, cryo transmission electron microscopy and atomic force microscopy were performed in the present study. |
3. (Line 69-73) I think that the reference (27) is not so recent. Please, rephrase the sentence. | That’s true. Based on the reviewer’s comment, the following description has been revised in the revised manuscript.
(Page 2, line 70) : Previously, we reported that the water-solubility of.... |
Comment #4
(Line 126) Please, supply the real temperature, the angle and the concentration of the formulations that were measured in DLS. How many measurements of each sample did the authors do?
Did the authors dilute the samples before the analysis?
Response:
Mean particle size, PDI and zeta potential were measured by zetasizer at a scattering angle of 90° at room temperature (25°C). In addition, the values of mean count rate of intact samples were 200–500 kcps which are suitable for this measurement; therefore, we did not perform the dilution in this study. And we repeated measurement three times per a sample. Based on the reviewer’s comment, the following sentence has been revised in the revised manuscript. |
:The mean particle size and the zeta potential of LN suspensions were measured with dynamic light scattering (DLS) (Zetasizer nano ZS, Malvern Instruments Ltd., Worcestershire, UK) at a scattering angle of 90° at room temperature (25°C). Mean particle sizes were based on the scattering intensity, while the zeta potential was based on electrophoretic mobility. Since the values of mean count rate of the samples were 200–500 kcps which are suitable for this measurement, intact samples without dilution were used in this measurement. We repeated measurement three times per a sample.
5. (Line 188) Liposomes are not particles. Liposomes are vesicles in nanoscale. Please, rephrase the sentence. | Thank you for your comment. Although we think that liposomes are one of lipid-based nanoparticles in a broad sense, as you pointed out, liposomes are definitely vesicles in nanoscale. Based on your comments, the following description has been revised in the revised manuscript. (Page 5, line 195) : Spherical vesicles such as liposomes sometimes strongly |
6. (line 206) How can the authors affirm that the mean particle size became slightly bigger after freeze-drying, but the bicelle structure was preserved”? Please, clarify it. In addition, the authors said that “Thus, freeze-drying and the addition of a lyophilization stabilizer did not affect the NI-LN structure”. How the lyophilization stabilizers did not affect the NI-LN structure if the particle size became bigger? For me it is not clear. How did the author measure the size relating to particles, micelles and bicelles as indicated inTable 1, 2 and 3? | Thank you for your comment and sorry for insufficient explanation. Firstly, as for the description (line 206), we judged this by just watching the Fig. 4B compared with Fig. 1 and 4A (just visual observation). But, after this description, we have already checked the mean particle size determined by cryo-TEM images (Table 1) and zetasizer using DLS (Table 2), and in fact, mean particle sizes were a little bit bigger than others. Based on the reviewer’s comments, this description seems to be confusable; so, the description has been revised as follows. (Page 6, line 214) : The particle size looked slightly bigger after freeze-drying, but the bicelle structure was preserved. An increase in particle size might be explained by insertion of sucrose molecules in the phospholipid molecules of the lipid bilayer. However, as shown in Fig. 4B, the disk structure of the bicelles was maintained after reconstitution. Namely, we cannot observe any structural changes from disk-like to spherical or other forms, for example. Therefore, we thought that freeze-drying and the addition of a lyophilization stabilizer did not affect the NI-LN structure itself. Actually, we have no idea why the particle size became slight bigger; but we speculate that some aggregation between comparatively big and small particles would occur. We hope that reviewer understand our thought. In addition, one hundred particles in cryo-TEM images printed on paper were measured with a ruler to determine the size relating to particles. So, the following description has been revised in the revised manuscript. Thank you for your comments. (Page 6, line 219) : one hundred particles in cryo-TEM images printed on paper were measured with a ruler (Table 1). |
7. (Line 271) Liposomes? Please, explain it for better understanding. | As shown in Fig. 6A and 6B, the shape did not change when the sample stage was rotated by 20 degrees; so the particles were thought not to be bicelles or flat particles. When looking at inner part of the particles, the particles were seems like to be liposomes having internal water phase because inner part looked transparent. If the particles were not liposomes, the inner part of the particles would look black. Furthermore, as we have already given the explanation (line 354-3555 in the original manuscript), it was reported that cholesterol-free liposomes became polygonal particle (#46 in the original manuscript). Therefore, we think this particle in nanoscale might be liposomes. Based on the reviewer’s comment, the same reference has been added in the revised manuscript. (Page 8, line 280) :Thus, the particles were not flat and could have been polygonal liposomes [47]. |
Author Response File: Author Response.pdf
Reviewer 3 Report
Dear Editor,
The paper by Matsuoa et al. entitled “Combination of roll grinding and high-pressure homogenization can prepare stable bicelles for drug delivery” comprehensively elucidates how these carriers improve the delivery some drugs. The subject fits to the journal scope and, in my opinion, is interesting for the scientific community.
This work provides an useful method for stable bicelles for drug delivery.
It needs a small revision of some parts. The enhanced solubility of water insoluble drugs seem to be interesting. Introduction part is well written and provided by proper references. The paper substantially deals with the characterization of these nanoparticles.
From the scientific point of view I have just one objection. Afterwards, only a formal issue.
Major request:
DLS measurements
The authors along the manuscript speak about the mean particle sizes based on the scattering intensity.
Please be a little more precise and clearer for researchers not dealing with DLS measurements. Specify that you are reading the Z-average, if I am true, and that this measure is speaking about the hydrodynamic radius or diameter of the particles and micelles. Please specify that usually this hydrodimanic radius is a little bigger than the particles itself by quoting the proper papers……. and so on.
For instance the sentences written from line 216 to line 218 and in line 288 are for sure correct but seems to me too simplistic.
Minor request:
LINE 177: “These changes indicated flat particles. “ (Please quote proper reference)
Based on the above arguments, I recommend the paper for publication.
Author Response
We thank the reviewers for the very valuable comments. We have addressed all the issues raised by the reviewers in a point-by-point manner as noted below. The changes made in the manuscript have been marked in red.
Reviewer #3. We thank the reviewer for his or her positive and useful comments.
Comments | Our response | |
1 | DLS measurements The authors along the manuscript speak about the mean particle sizes based on the scattering intensity. Please be a little more precise and clearer for researchers not dealing with DLS measurements. Specify that you are reading the Z-average, if I am true, and that this measure is speaking about the hydrodynamic radius or diameter of the particles and micelles. Please specify that usually this hydrodimanic radius is a little bigger than the particles itself by quoting the proper papers……. and so on. For instance…. the sentences written from line 216 to line 218 and in line 288 are for sure correct but seems to me too simplistic. | Thank you so much for your comments. As you pointed out, it is known that mean particle size measured by scattering intensity from brownian motion is bigger than that of measured by other methods. Because bigger particles can influence scattering intensity, DLS has reported to tend to overestimate the hydrodynamic diameter than real one (Magdalena, W. et al., Colloids and Surfaces B: Biointerfaces 2014). Moreover, DLS furnishes the hydrodynamic diameter depending on assumption that the particle is sphere (Doroty, C. et al., Int. J. Pharm. 2015), but the bicelles in this study were not spherical; so we thought the possibility that the data from DLS might be not accurate. Thus, we think measurement by only DLS seems not to be sufficient and measurement by cryo-TEM would also be necessary. Based on the reviewer’s comments, the following descriptions have been revised and new references have been added to the revised manuscript. (Page 6, line 218) :Cryo-TEM and DLS data were based on the number reference and on scattering intensity from brownian motion, respectively. Previously, it was reported that DLS tends to overestimate the hydrodynamic diameter than real one because large particles strongly influences scattering intensity [44]. In addition, DLS furnished the hydrodynamic diameter depending on assumption that the particles are sphere [45], but the bicelles in this study were not spherical. Therefore, the difference of measurement principle would be involved in the differences of particle size. (References) 44. Magdalena, W.; Jan, B.; Maria, N.; Paweł, W.; Mariusz, K. Interactions of serum with polyelectrolyte-stabilized liposomes: Cryo-TEM studies. Colloids and Surfaces B: Biointerfaces 2014, 120, 152-159. 45. Doroty, C.; Jonathan, C.; Jenna, B.; William, J. M.; Maria, A. O.; Sheng, Q. Disc-shaped polyoxyethylene glycol glycerides gel nanoparticles as novel protein delivery vehicles. Int. J. Pharm. 2015, 496, 1015-1025. |
2 | LINE 177: “These changes indicated flat particles. “ (Please quote proper reference) | Thank you for your comment. The following description has been revised and new reference has been added in the revised manuscript. (Page 4, line 184) :These changes indicated flat particles as previously reported [39] (Reference) 39. Yasuhara, K.; Miki, S.; Nakazono, H.; Ohta, A.; Kikuchi, J. Synthesis of organic–inorganic hybrid bicelles–lipid bilayer nanodiscs encompassed by siloxane surfaces.Chem. Commun., 2011, 47, 4691–4693. |
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
The authors properly discussed the proposed crticisms in the revised version.
The manuscript is worthy of publication.
Reviewer 3 Report
Dear editor,
the authors have done a great job answering correctly all my criticisms and by adding proper references.
Please accept in this form.
