Development of Paclitaxel Proliposomal Dry Powder Inhaler (PTX-PLM-DPI) by Freeze-Drying Method for Lung Cancer

Round 1

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The authors have considered feedback from reviewers and revised the manuscript based on that input.

A few areas are still not adequately addressed and the authors must consider  the problems in the manuscript and address them.

Page 2line 51:  authors state that "Most DPIs are prepared by spray drying methods..." .I believe the vast majority of DPIs are made by  blending micronized drug substance with fine lactose powder and coarser lactose powder.  Please provide detailed evidence to support the statement about most DPIs being prepared by spray drying. I do agree that the two examples  provided (tobramycin and mannitol) in the revision are spray dried powders for inhalation.

 

Page 7 line 286- 290: Figure 1 shows the paclitaxel proliposome DPI powder under the SEM pre and post lyophilisation. Apart from the degree of apparent aggregation which is likely associated with sample preparation, the images look similar. Authors might wish to comment on differences in  surface structure.

Additionally these images suggest  individual particles of mannitol (I assume that these are the selected mannitol formulation and not the lactose monohydrate formulation) are all quite large, many tens and approaching the hundreds of micrometres, at least based on the scale bar given.  How can this observation be reconciled with the delivery of a fine particle fraction (less than/equal to 5 micrometres) being about 50% of the amount of drug deposited Page 10 line 342).  Is the total amount of fine particle fraction drug deposited  just a fraction of the  drug delivered?  So is the remainder of the cytotoxic drug lodged higher up in the respiratory tract? Is that a safety issue for patients?   Please discuss further in Page 10 lines 344 - 349, the relationship between  weight of formulation  emitted from the capsule and the weight deposited in the stages of the impactor.  There is something odd going on here between the carrier particles size and what arrives in the impactor.  Have you a particle size distribution of the  carrier used?

Page 10 line 342:  when you are calculating delivered drug mean amount, I do not think you can determine that to five decimal places of micrograms! Use the number of decimal places aligned with the sensitivity of the balance used to weigh the materials delivered (likely one decimal place of micrograms?). Please check for this issue throughout the entire manuscript.

 

 

Author Response

1. Page 2 line 51: authors state that "Most DPIs are prepared by spray drying methods..."I believe the vast majority of DPIs are made by blending micronized drug substance with fine lactose powder and coarser lactose powder. Please provide detailed evidence to support the statement about most DPIs being prepared by spray drying. I do agree that the two examples  provided (tobramycin and mannitol) in the revision are spray dried powders for inhalation.

Response: Thank you for your comment. We do agree that the vast majority of DPIs are made by blending micronized drug substance with fine lactose powder and coarser lactose powder. So, in the revised version of the manuscript, we have replaced the word “most” with “In some cases, DPIs are prepared by spray drying method”. Kindly note that modified text has been highlighted in blue color.

 

2. Page 7 line 286- 290: Figure 1 shows the paclitaxel proliposome DPI powder under the SEM pre and post lyophilisation. Apart from the degree of apparent aggregation which is likely associated with sample preparation, the images look similar. Authors might wish to comment on differences in surface structure.

Response: Thank you for your comment. Apart from the degree of apparent aggregation which is likely associated with sample preparation, the images are indeed similar. There is no significant morphological differences pre-and post-lyophilisation of the DPIs. However, significant improvement of flow property has been observed post lyophilisation.

 

3. Additionally these images suggest individual particles of mannitol (I assume that these are the selected mannitol formulation and not the lactose monohydrate formulation) are all quite large, many tens and approaching the hundreds of micrometres, at least based on the scale bar given. How can this observation be reconciled with the delivery of a fine particle fraction (less than/equal to 5 micrometres) being about 50% of the amount of drug deposited Page 10 line 342). Is the total amount of fine particle fraction drug deposited just a fraction of the drug delivered?  So is the remainder of the cytotoxic drug lodged higher up in the respiratory tract? Is that a safety issue for patients?   Please discuss further in Page 10 lines 344 - 349, the relationship between weight of formulation emitted from the capsule and the weight deposited in the stages of the impactor.  There is something odd going on here between the carrier particles size and what arrives in the impactor.  Have you a particle size distribution of the  carrier used?

Response: Thank you for your insightful remark. SEM images suggest carrier particles of proliposomes within the size range of 50-80 µm which are apparently very large. While during in vitro lung deposition study using the cascade impactor a certain amount of suction force is applied which detaches the proliposomal particle from the carrier and deposited at the pre separator. Since the carrier particle is been detached, the proliposome distributes through the different stages (0-7) assembly of the cascade impactor in terms of its original size (0.5-9 µm). The particle deposition was more at pre separator and distributed up to stage 7 indicating that particles were able to pass up to the lowest stage in sufficient quantity. The fine particle fraction (FPF) (aerodynamic diameter less than 5µ) deposited in stage 2 to 7 was found to be 50.86±2.8%.  In Vitro Aerosol Performance Parameters of the dry powder formulation determined with an Anderson Cascade Impactor is attached here for your reference:

ACI stage	Particle size (µm)	Amount of drug deposited (µg)±SD
Device		26.17± 2.44
Preseparator		103.99± 0.11
Stage 0	9.0+	78.13± 4.55
Stage 1	7.4	34.80± 3.47
Stage 2	5.25	68.34± 1.98
Stage 3	4	35.64± 4.07
Stage 4	2.7	42.99± 3.66
Stage 5	1.6	35.15± 2.13
Stage 6	0.9	25.70± 6.01
Stage 7	0.55	25.36± 5.66

 

4. Page 10 line 342: when you are calculating delivered drug mean amount, I do not think you can determine that to five decimal places of micrograms! Use the number of decimal places aligned with the sensitivity of the balance used to weigh the materials delivered (likely one decimal place of micrograms?). Please check for this issue throughout the entire manuscript.

Response: Thank you for your valuable comment. While calculating delivered drug mean amount, we did not use weighing balance, instead we quantified the delivered drug amount using HPLC. After the aerosolization tests, the ACI was dismantled and the powder collected at various stages was quantified in ethanol using Waters HPLC consisting of UV detector at 230 nm and a flow rate of 1 mL/min. Acetonitrile and water was used as the mobile phase at 75:25 v/v. The mass median aerodynamic diameter (MMAD) was determined, which is the aerodynamic diameter below which 50 % of particles remain. The fine particle fraction (FPF) was determined using the following formula [9]:

FPF= (Drugs in stages 2-7)/ (Drugs in all stages)×100%

 

 

 

 

 

 

 

 

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

The authors tried to improve the quality however, it still needs significant improvement. I would recommend going through the manuscript consciously and edit accordingly. Some major comments are here:

The formulated LOD and LOQ formulas are wrong. What were the LOD and LOQ values authors determined? Mention in the manuscript.

The entrapment efficiency formula is not correct (it is wrong in the referenced article too). I would recommend adding another reference.

Figure 2: divide in A, and B sections, mention the scalebar in the caption. The quality of the DLS graph is not good. Mention PDI value and average DLS size.

Author Response

1. The formulated LOD and LOQ formulas are wrong. What were the LOD and LOQ values authors determined? Mention in the manuscript.

Response: Thank you for your insightful remark. We are extremely sorry for the incorrect LOD and LOQ formulas. However, we did not determine the LOD and LOQ as we are using a previously developed method. Hence, we have removed the LOD and LOQ from the revised version of the manuscript.

 

2. The entrapment efficiency formula is not correct (it is wrong in the referenced article too). I would recommend adding another reference.

Response: Thank you for your insightful remark. As per your suggestion, we have corrected the entrapment efficiency formulated in the revised version of the manuscript. Kindly note that modified text has been highlighted in blue color.

 

3. Figure 2: divide in A, and B sections, mention the scalebar in the caption. The quality of the DLS graph is not good. Mention PDI value and average DLS size.

Response: Thank you for your comment. As per your suggestion, we have modified and divided figure 2 into section A, B and C and also added the scalebar. The quality of the DLS graph has been improved alongwith mentioning PDI value and average DLS size.

Author Response File: Author Response.pdf

Reviewer 3 Report (New Reviewer)

Comments and Suggestions for Authors

This manuscript describes the development of a Paclitaxel encapsulated powder.  The materials are extensively characterized.  The applications well thought out and tested.

All of the edits made by the authors of this manuscript are reasonable, and improve the manuscript.  

 

Author Response

This manuscript describes the development of a Paclitaxel encapsulated powder.  The materials are extensively characterized.  The applications well thought out and tested.

All of the edits made by the authors of this manuscript are reasonable, and improve the manuscript.  

Response: Thank you very much for your appreciation and positive response.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

The manuscript still requires further improvement to  clearly describe and present the research done and its findings. In particular the dose delivered and the lack of connect between the particle size of the carrier and the  fine particle fraction delivered is very problematic and needs clarifying.

In their response to the last review feedback the authors state:

"SEM images suggest carrier particles of proliposomes within the size range of 50-80 µm which are apparently very large. While during in vitro lung deposition study using the cascade impactor a certain amount of suction force is applied which detaches the proliposomal particle from the carrier and deposited at the pre separator. Since the carrier particle is been detached, the proliposome distributes through the different stages (0-7) assembly of the cascade impactor in terms of its original size (0.5-9 µm). The particle deposition was more at pre separator and distributed up to stage 7 indicating that particles were able to pass up to the lowest stage in sufficient quantity. The fine particle fraction (FPF) (aerodynamic diameter less than 5µ) deposited in stage 2 to 7 was found to be 50.86±2.8%"

Where in the SEM images can we see the proliposomal particles they describe as detaching to create the fine particle fraction? Are they saying they are created in the simulated inhalation process of the impactor? Please make this clear. Also that 50.86% fine particle fraction, please indicate somewhere in the appropriate section what  percentage of the drug contained n the capsule  is in this fine particle fraction.  Understanding how much is not delivered will be key to  clarifying how much of the drug in the capsule gets  generated into fine particles. I assume it is very small as it is created by an attrition process it would seem.

 

Author Response

Thank you for your insightful remark. The SEM study was conducted to check the surface morphology of the PLM. We cannot see the proliposomal particle detaching to create the fine particle fraction. The surface morphology of pre and post lyophilized PLM showed no major differences and showed formation of small, uniform size nanoparticles absorbed on the surface of the carriers with no sign of aggregation (Figure 1a and b).

            The drug content in the capsule in the fine particle section has been mentioned in the section: In vitro Lung Deposition Study. Kindly refer to page 6.

            Proliposomal are not created in the simulated inhalation process of the impactor. During the simulation process of the Anderson Cascade Impactor, the proliposomal particles detaches from the carrier and gets deposited at the pre separator.

            The total proliposomal powder weight in the capsule was 25 mg whereas the paclitaxel content was            = Theoretical content of paclitaxel* 95.6 %

= 500 µg * 95.6% (92% is the drug entrapment in the formulation. Kindly refer       

                 to page 5, section : Entrapment efficiency and in vitro drug release)

= 478 µg

This 478 µg (Paclitaxel content in the capsule) aligns with the results obtained from the cascade impactor as provided in table 4.

Out of this 478 µg drug, 50.86 % drug lies between phase 2 to phase 7 while performing the in vitro lung deposition study using the cascade impactor.
              The detailed procedure of the in vitro lung deposition study is given below:
25 mg of dry powder formulation was packed into three hard gelatin capsules (size 3) in a laboratory setting. The capsules were taken in the Rotahaler® inhaler device and attached to the induction port with the help of a rubber mouthpiece adapter. Using a Rotahaler® inhaler device, the formulation was actuated into the ACI at a consistent flow rate of 60 L/min for every experiment. After the aerosolization tests, the ACI was dismantled. The amount of fine particles collected in each stage was insufficient to determine their weight and the amount of drug collected at various stages was quantified in ethanol using Waters HPLC consisting of UV detector at 230 nm and a flow rate of 1 mL/min. Acetonitrile and water was used as the mobile phase at 75:25 v/v. The mass median aerodynamic diameter (MMAD) was determined, which is the aerodynamic diameter below which 50 % of particles remain. The fine particle fraction (FPF) was determined using the following formula:

 

            Total percentage drug delivered in the lung = (233/478)* 100%

                                                                = 48.7 %

[273 µg is the amount of drug between phase 2 to phase 7 whereas 478 µg is the total drug content/entrapment efficiency of the proliposomal powder.

Please note that the total drug in the in viro lung deposition study, right from device, preseparator, from phase 0 to phase 7 is 473 µg (26.17+ 103.99+ 78.13 +34.80 + 68.34 + 1.98

+ 35.64 + 42.99 + 35.15+ 25.70 +25.36 µg) ]

Author Response File: Author Response.pdf

Reviewer 2 Report (Previous Reviewer 2)

Comments and Suggestions for Authors

Figures 1 and 2: Add scale bar values in the caption. example (Scale 20 uM)

Author Response

1. Figures 1 and 2: Add scale bar values in the caption. example (Scale 20 uM)

Response: Thank you for your valuable suggestion. As per your suggestion, we have added scale bar values in figure 1 and 2.

 

Author Response File: Author Response.pdf

Round 3

Reviewer 1 Report (Previous Reviewer 1)

Comments and Suggestions for Authors

I am still not happy with the description of the origin of the fine particle fraction, particularly having issue with  the SEM images and the disconnect with the generation of 50% approx. by mass of fine particle fraction from the powder.  Authors have said:

"The SEM study was conducted to check the surface morphology of the PLM. We cannot see the proliposomal particle detaching to create the fine particle fraction. The surface morphology of pre and post lyophilized PLM showed no major differences and showed formation of small, uniform size nanoparticles absorbed on the surface of the carrier..."

I cannot easily discern any fines in the SEM images, just  a lot of large crystals and no clear evidence of uniform size nanoparticles adsorbed on the surface of the crystals. Authors should consider higher magnification images of carrier particles to show the nanoparticles on the surface and  run a particle size analysis preferably by laser light scattering method to capture  very small 0.5- 10 micron particles, analysing the freeze dried powder to  show the presence of significant level of single digit micron particles.  Then  the text of the manuscript should be   added to to describe the origin of the fine particle fraction.

Author Response

Comment: I am still not happy with the description of the origin of the fine particle fraction, particularly having issue with the SEM images and the disconnect with the generation of 50% approx. by mass of fine particle fraction from the powder.  Authors have said:

"The SEM study was conducted to check the surface morphology of the PLM. We cannot see the proliposomal particle detaching to create the fine particle fraction. The surface morphology of pre and post lyophilized PLM showed no major differences and showed formation of small, uniform size nanoparticles absorbed on the surface of the carrier..."

I cannot easily discern any fines in the SEM images, just a lot of large crystals and no clear evidence of uniform size nanoparticles adsorbed on the surface of the crystals. Authors should consider higher magnification images of carrier particles to show the nanoparticles on the surface and  run a particle size analysis preferably by laser light scattering method to capture  very small 0.5- 10 micron particles, analysing the freeze dried powder to  show the presence of significant level of single digit micron particles.  Then the text of the manuscript should be   added to describe the origin of the fine particle fraction.

Response: Thank you for your insightful remark. We have tried to highlight the adsorbed particles on the surface of the carriers as shown in the SEM image of higher magnification (Please find attached SEM image in the word file). Please note that the size of DPI are in micron range, not in nano range (as shown in SEM image) which is essential for their alveolar deposition. For size determination of these freeze dried detached particles, Anderson Cascade Impactor was used that has been reported in the manuscript. In the cascade impactor which is equipped with filters of decreasing pore size the DPI derived fine particles gets separated. Based on the pore size of the filters (Stage 0: 9.0+ µm; Stage 1: 7.40 µm; Stage 2: 5.25 µm; Stage 3: 4.00 µm; Stage 4: 2.70 µm; Stage 5: 1.60 µm: Stage 6: 0.90 µm; Stage 7: 0.55 µm) the fine particles were collected in different plates which were then quantified by HPLC after dispersing in Acetonitrile. As has been reported the particles were collected upto stage 7 whose pore size is 0.55 µm. Please note that, these proliposmes on dispersion with aqueous phase gets converted to liposomes which are in nanometer range and has been evaluated by DLS method and the sizes have been reported. The size of optimised liposomes obtained from reconstitution of these proliposome found to be 196±5 nm. This nanorange image of the optimized liposomes reconstituted from the proliposomes have been further analysed by the TEM analysis which also shows similar size particles less than 200 nm.

 

 

 

Author Response File: Author Response.pdf

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have explored a proliposome approach to enable the delivery of paclitaxel to the lungs.

The work may be of relevance to the scope of the journal in consideration for publication but there are aspects of the manuscript that need further  effort by the authors before the manuscript should be  considered further for publication.

 

Page 1 line 32: please provide reference to support the claim around the use of liposome formulations as the most successful  approach in targeted cancer therapy.  The reference [1] cited is not a clinical study.

Page 1 line 37-40 introduces the concept  of proliposomes without properly describing what they are and does not provide the primary  literature appearances of the technology and terminology. Reference 3-4  were published   some 30 or more years later post the first publication of the concept.

Page 2, line 48: notes that most DPIs are made by spray drying. Please provide examples of  commercial products made by this technology. I believe most DPIs are made by dry powder blending of crystalline (usually so) materials notably controlled particle size lactose as a carrier.

Page 2, line51: my experiences with proliposomes  suggest the sticky phospholipid coating around the saccharide carrier impairs flow and can cause aggregation.  How did the authors  manipulate their composition and process to manage this?

Page 2, line 95:  what was the particle size grade of the lactose and of the mannitol used as carriers?  Correct spelling of acetonitrile.

Page 3, line 105:  authors use chloroform to prepare the proliposomes.  Chloroform is a "solvent to be limited" in pharmaceutical products under the ICH Q3c guidance with a strict control of 60 ppm in the finished product.  How did authors assure control of very low levels of chloroform in their proliposomes?

Page 4 lines 185-186: why is pH 7.4 phosphate buffered saline with 0.05% SLS considered an appropriate medium to simulate alveolar fluid?

Page 6 line 259: I could not discern small uniform size nanoparticles in the images in figures 1a ad 1b. Please provide higher magnification images to illustrate this.

Page 6 figure 1a and Page 7 figure 1b:  SEM images suggest carrier particles that provide for in situ generation of liposomes from proliposomes appear very large (crystals appear to be 50 - 80 micrometres in length).  This does not tally with the cascade impactor data in Table 4. Please provide explanation of lack of correlation.

Page 7, line 276-279: whilst you might expect that the crystallinity of PTX might be lost due dispersion in phospholipid, could the inability to  discern the peaks for crystalline PTX also be that it is diluted out and swamped by the  PXRD pattern of the carrier?

Page 10 line 336, Table 4:  This data appears odd considering the very large relatively uniform crystalline particles seen in Fig 1b. Is the cascade impactor data reliable?  What does the size distribution look like if you just put the mannitol carrier through the impactor test?

Page 11 Figure 5: this is unnecessary if we have the numerical data  in table 4. In any case the stages in the figure should have the particle size they relate to.

Reviewer 2 Report

Comments and Suggestions for Authors

The study is interesting however, I would recommend a major revision of the manuscript before publication.

Methods:

·         Line 108-109: The carbohydrate carrier (Mannitol or LMH) was mixed to the lipid phase in different ratios.->> Mention proper ratios. (also check the grammar in this sentence).

·         Line 110-111: PLMs 110 were then collected, lyophilized, and stored: ->> Here the preparation step of PLM is missing. Explain the proper synthetic procedure.

·         How the liposomes were characterized before and after lyophilization is missing. Explain.

·         In Hausner's ratio and Car’s index determination, what was the volume of the measuring cylinder? Until how much volume before tapping was filled? What was the force of tapping and how many times/until when the cylinder was tapped after filling? Explain the procedure properly.

·         Line 134-135: The powder samples were scattered across aluminium microscope stubs of the apparatus and painted with goldà Authors did not use carbon tape, how are the aluminium stubs painted with gold? What about samples of gold coating? This does not seem like a proper sample preparation. I would suggest to write properly.

·         Line 145-146: what was the ultrasonicator instrument capacity, model, and make? At what energy the sample was sonicated? 30 min sonication is quite high, does it not affect the encapsulation efficiency?

·         Line 170-171: Mention the LOD and LOQ equations in this section.

·         The entrapment efficiency determination procedure seems incorrect. The first question is, how, the un-entrapped drug can be in the supernatant since it is water-insoluble? Second, it is impossible to settle down liposomes at 5000 rpm in 10 min which is a quite low speed. Then how entrapment efficiency was determined in the supernatant?? Moreover, the efficiency formula applied here is wrong, check it again with other literature.

Results:

·         How the 95.26% w/w yield of PTX-PLM-DPI was calculated?

·         Merge figures 1a and b in a single figure.

·         In the TEM figure a lot of aggregation can be observed, if possible add more TEM images.

·         It is important to add DLS graphs with the TEM images to confirm the monodispersity and intensity of the particles. I would recommend adding these figures.

·         Figure 3: what is the upper figure and lower figure? Name (a) and (b) and mention them in the caption. Moreover, the quality of the figure is not good increase the axis major tick fonts and titles.  Improve the quality of the figure.

·         Figures 5 and 6: there is no comparison between various groups in these figures to determine the efficacy. I would recommend adding a comparative ANOVA analysis with a suitable post hoc test. Moreover, increase the quality of the figures.

Others

·         The units throughout the manuscript must be uniform. For example: change ml to mL, l to L throughout.

·         Check grammar throughout

Comments on the Quality of English Language

Check Grammar