Targeted drug delivery to the skin and deeper tissues: Role of physiology, solute structure and disease

1. Drug delivery through the skin has been used to target the epidermis, dermis and deeper tissues and for systemic delivery, The major barrier for the transport of drugs through the skin is the stratum corneum, with most transport occurring through the intercellular region, The polarity of the intercellular region appears to be similar to butanol, with the diffusion of solutes being hindered by saturable hydrogen bonding to the polar head groups of the ceramides, fatty acids and other intercellular lipids, Accordingly, the permeability of the more lipophilic solutes is greatest from aqueous solutions, whereas polar solute permeability is favoured by hydrocarbon-based vehicles. 2. The skin is capable of metabolizing many substances and, through its microvasculature, limits the transport of most substances into regions below the dermis. 3. Although the flux of solutes through the skin should be identical for different vehicles when the solute exists as a saturated solution, the fluxes vary in accordance with the skin penetration enhancement properties of the vehicle. It is therefore desirable that the regulatory standards required for the bioequivalence of topical products include skin studies. 4. Deep tissue penetration can be related to solute protein binding, solute molecular size and dermal blood flow. 5. Iontophoresis is a promising area of skin drug delivery, especially for ionized solutes and when a rapid effect is required. 6. In general, psoriasis and other skin diseases facilitate drug delivery through the skin. 7. It is concluded that the variability in skin permeability remains an obstacle in optimizing drug delivery by this route.

Scale Up of PLA Nanoparticle Synthesis by Solvent Displacement and Functionalization of the Particles with a Silica Shell for Targeted Drug Delivery Applications

Biodegradable polymer nanoparticles have the properties necessary to address many of the issues associated with current drug delivery techniques including targeted and controlled delivery. A novel drug delivery vehicle is proposed consisting of a poly(lactic acid) nanoparticle core, with a functionalized, mesoporous silica shell. In this study, the production of PLA nanoparticles is investigated using solvent displacement in both a batch and continuous manner, and the effects of various system parameters are examined. Using Pluronic F-127 as the stabilization agent throughout the study, PLA nanoparticles are produced through solvent displacement with diameters ranging from 200 to 250 nm using two different methods: dropwise addition and in an impinging jet mixer. The impinging jet mixer allows for easy scale-up of particle production. The concentration of surfactant and volume of quench solution is found to have minimal impact on particle diameter; however, the concentration of PLA is found to significantly impact the diameter mean and polydispersity. In addition, the stability of the PLA nanoparticles is observed to increase as residual THF is evaporated. Lastly, the isolated PLA nanoparticles are coated with a silica shell using the Stöber Process. It is found that functionalizing the silica with a phosphonic silane in the presence of excess Pluronic F-127 decreases coalescence of the particles during the coating process. Future work should be conducted to fine-tune the PLA nanoparticle synthesis process by understanding the effect of other system parameters and in synthesizing mesoporous silica shells.

Magnetic nanoparticle-based targeted drug delivery for treatment of neuro-AIDS and drug addiction

Brain is one of the safe sanctuaries for HIV and, in turn, continuously supplies active viruses to the periphery. Additionally, HIV infection in brain results in several mild-to-severe neuro-immunological complications termed neuroAIDS. One-tenth of HIV-infected population is addicted to recreational drugs such as opiates, alcohol, nicotine, marijuana, etc. which share common target-areas in the brain with HIV. Interestingly, intensity of neuropathogenesis is remarkably enhanced due to exposure of recreational drugs during HIV infection. Current treatments to alleviate either the individual or synergistic effects of abusive drugs and HIV on neuronal modulations are less effective at CNS level, basically due to impermeability of therapeutic molecules across blood-brain barrier (BBB). Despite exciting advancement of nanotechnology in drug delivery, existing nanovehicles such as dendrimers, polymers, micelles, etc. suffer from the lack of adequate BBB penetrability before the drugs are engulfed by the reticuloendothelial system cells as well as the uncertainty that if and when the nanocarrier reaches the brain. Therefore, in order to develop a fast, target-specific, safe, and effective approach for brain delivery of anti-addiction, anti-viral and neuroprotective drugs, we exploited the potential of magnetic nanoparticles (MNPs) which, in recent years, has attracted significant importance in biomedical applications. We hypothesize that under the influence of external (non-invasive) magnetic force, MNPs can deliver these drugs across BBB in most effective manner. Accordingly, in this dissertation, I delineated the pharmacokinetics and dynamics of MNPs bound anti-opioid, anti-HIV and neuroprotective drugs for delivery in brain. I have developed a liposome-based novel magnetized nanovehicle which, under the influence of external magnetic forces, can transmigrate and effectively deliver drugs across BBB without compromising its integrity. It is expected that the developed nanoformulations may be of high therapeutic significance for neuroAIDS and for drug addiction as well.

Magnetic Nanoparticle-based Targeted Drug Delivery for Treatment of Neuro-AIDS and Drug Addiction

Brain is one of the safe sanctuaries for HIV and, in turn, continuously supplies active viruses to the periphery. Additionally, HIV infection in brain results in several mild-to-severe neuro-immunological complications termed neuroAIDS. One-tenth of HIV-infected population is addicted to recreational drugs such as opiates, alcohol, nicotine, marijuana, etc. which share common target-areas in the brain with HIV. Interestingly, intensity of neuropathogenesis is remarkably enhanced due to exposure of recreational drugs during HIV infection. Current treatments to alleviate either the individual or synergistic effects of abusive drugs and HIV on neuronal modulations are less effective at CNS level, basically due to impermeability of therapeutic molecules across blood-brain barrier (BBB). Despite exciting advancement of nanotechnology in drug delivery, existing nanovehicles such as dendrimers, polymers, micelles, etc. suffer from the lack of adequate BBB penetrability before the drugs are engulfed by the reticuloendothelial system cells as well as the uncertainty that if and when the nanocarrier reaches the brain. Therefore, in order to develop a fast, target-specific, safe, and effective approach for brain delivery of anti-addiction, anti-viral and neuroprotective drugs, we exploited the potential of magnetic nanoparticles (MNPs) which, in recent years, has attracted significant importance in biomedical applications. We hypothesize that under the influence of external (non-invasive) magnetic force, MNPs can deliver these drugs across BBB in most effective manner. Accordingly, in this dissertation, I delineated the pharmacokinetics and dynamics of MNPs bound anti-opioid, anti-HIV and neuroprotective drugs for delivery in brain. I have developed a liposome-based novel magnetized nanovehicle which, under the influence of external magnetic forces, can transmigrate and effectively deliver drugs across BBB without compromising its integrity. It is expected that the developed nanoformulations may be of high therapeutic significance for neuroAIDS and for drug addiction as well.

Review of therapeutic drug monitoring of anticancer drugs part two - Targeted therapies.

Most of oral targeted therapies are tyrosine kinase inhibitors (TKIs). Oral administration generates a complex step in the pharmacokinetics (PK) of these drugs. Inter-individual PK variability is often large and variability observed in response is influenced not only by the genetic heterogeneity of drug targets, but also by the pharmacogenetic background of the patient (e.g. cytochome P450 and ABC transporter polymorphisms), patient characteristics such as adherence to treatment and environmental factors (drug-drug interactions). Retrospective studies have shown that targeted drug exposure, reflected in the area under the plasma concentration-time curve (AUC) correlates with treatment response (efficacy/toxicity) in various cancers. Nevertheless levels of evidence for therapeutic drug monitoring (TDM) are however heterogeneous among these agents and TDM is still uncommon for the majority of them. Evidence for imatinib currently exists, others are emerging for compounds including nilotinib, dasatinib, erlotinib, sunitinib, sorafenib and mammalian target of rapamycin (mTOR) inhibitors. Applications for TDM during oral targeted therapies may best be reserved for particular situations including lack of therapeutic response, severe or unexpected toxicities, anticipated drug-drug interactions and/or concerns over adherence treatment. Interpatient PK variability observed with monoclonal antibodies (mAbs) is comparable or slightly lower to that observed with TKIs. There are still few data with these agents in favour of TDM approaches, even if data showed encouraging results with rituximab, cetuximab and bevacizumab. At this time, TDM of mAbs is not yet supported by scientific evidence. Considerable effort should be made for targeted therapies to better define concentration-effect relationships and to perform comparative randomised trials of classic dosing versus pharmacokinetically-guided adaptive dosing.

Synthesis and Biological Evaluation of Novel Folic Acid Receptor-Targeted, β-Cyclodextrin-Based Drug Complexes for Cancer Treatment

Drug targeting is an active area of research and nano-scaled drug delivery systems hold tremendous potential for the treatment of neoplasms. In this study, a novel cyclodextrin (CD)-based nanoparticle drug delivery system has been assembled and characterized for the therapy of folate receptor-positive [FR(+)] cancer. Water-soluble folic acid (FA)-conjugated CD carriers (FACDs) were successfully synthesized and their structures were confirmed by 1D/2D nuclear magnetic resonance (NMR), matrix-assisted laser desorption ionization time-of-flight mass spectrometer (MALDI-TOF-MS), high performance liquid chromatography (HPLC), Fourier transform infrared spectroscopy (FTIR), and circular dichroism. Drug complexes of adamatane (Ada) and cytotoxic doxorubicin (Dox) with FACD were readily obtained by mixed solvent precipitation. The average size of FACD-Ada-Dox was 1.5–2.5 nm. The host-guest association constant Ka was 1,639 M−1 as determined by induced circular dichroism and the hydrophilicity of the FACDs was greatly enhanced compared to unmodified CD. Cellular uptake and FR binding competitive experiments demonstrated an efficient and preferentially targeted delivery of Dox into FR-positive tumor cells and a sustained drug release profile was seen in vitro. The delivery of Dox into FR(+) cancer cells via endocytosis was observed by confocal microscopy and drug uptake of the targeted nanoparticles was 8-fold greater than that of non-targeted drug complexes. Our docking results suggest that FA, FACD and FACD-Ada-Dox could bind human hedgehog interacting protein that contains a FR domain. Mouse cardiomyocytes as well as fibroblast treated with FACD-Ada-Dox had significantly lower levels of reactive oxygen species, with increased content of glutathione and glutathione peroxidase activity, indicating a reduced potential for Dox-induced cardiotoxicity. These results indicate that the targeted drug complex possesses high drug association and sustained drug release properties with good biocompatibility and physiological stability. The novel FA-conjugated β-CD based drug complex might be promising as an anti-tumor treatment for FR(+) cancer.

Therapeutic drug monitoring in cancer - Are we missing a trick?

Therapeutic drug monitoring (TDM) can be defined as the measurement of drug in biological samples to individualise treatment by adapting drug dose to improve efficacy and/or reduce toxicity. The cytotoxic drugs are characterised by steep dose-response relationships and narrow therapeutic windows. Inter-individual pharmacokinetic (PK) variability is often substantial. There are, however, a multitude of reasons why TDM has never been fully implemented in daily oncology practice. These include difficulties in establishing appropriate concentration target, common use of combination chemotherapies and the paucity of published data from pharmacological trials. The situation is different with targeted therapies. The large interindividual PK variability is influenced by the pharmacogenetic background of the patient (e.g. cytochrome P450 and ABC transporters polymorphisms), patient characteristics such as adherence to treatment and environmental factors (drug-drug interactions). Retrospective studies have shown that targeted drug exposure correlates with treatment response in various cancers. Evidence for imatinib currently exists, others are emerging for compounds including nilotinib, dasatinib, erlotinib, sunitinib, sorafenib and mammalian target of rapamycin (mTOR) inhibitors. Applications for TDM during oral targeted therapies may best be reserved for particular situations including lack of therapeutic response, severe or unexpected toxicities, anticipated drug-drug interactions and concerns over adherence treatment. There are still few data with monoclonal antibodies (mAbs) in favour of TDM approaches, even if data showed encouraging results with rituximab and cetuximab. TDM of mAbs is not yet supported by scientific evidence. Considerable effort should be made for targeted therapies to better define concentration-effect relationships and to perform comparative randomised trials of classic dosing versus pharmacokinetically-guided adaptive dosing.

Lethal weapons : novel approaches for receptor-targeted cancer cell elimination

The currently used forms of cancer therapy are associated with drug resistance and toxicity to healthy tissues. Thus, more efficient methods are needed for cancer-specific induction of growth arrest and programmed cell death, also known as apoptosis. Therapeutic forms of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) are investigated in clinical trials due to the capability of TRAIL to trigger apoptosis specifically in cancer cells by activation of cell surface death receptors. Many tumors, however, have acquired resistance to TRAIL-induced apoptosis and sensitizing drugs for combinatorial treatments are, therefore, in high demand. This study demonstrates that lignans, natural polyphenols enriched in seeds and cereal, have a remarkable sensitizing effect on TRAIL-induced cell death at non-toxic lignan concentrations. In TRAIL-resistant and androgen-dependent prostate cancer cells we observe that lignans repress receptor tyrosine kinase (RTK) activity and downregulate cell survival signaling via the Akt pathway, which leads to increased TRAIL sensitivity. A structure-activity relationship analysis reveals that the γ-butyrolactone ring of the dibenzylbutyrolactone lignans is essential for the rapidly reversible TRAIL-sensitizing activity of these compounds. Furthermore, the lignan nortrachelogenin (NTG) is identified as the most efficient of the 27 tested lignans and norlignans in sensitization of androgen-deprived prostate cancer cells to TRAIL-induced apoptosis. While this combinatorial anticancer approach may leave normal cells unharmed, several efficient cancer drugs are too toxic, insoluble or unstable to be used in systemic therapy. To enable use of such drugs and to protect normal cells from cytotoxic effects, cancer-targeted drug delivery vehicles of nanometer scale have recently been generated. The newly developed nanoparticle system that we tested in vitro for cancer cell targeting combines the efficient drug-loading capacity of mesoporous silica to the versatile particle surface functionalization of hyperbranched poly(ethylene imine), PEI. The mesoporous hybrid silica nanoparticles (MSNs) were functionalized with folic acid to promote targeted internalization by folate receptor overexpressing cancer cells. The presented results demonstrate that the developed carrier system can be employed in vitro for cancer selective delivery of adsorbed or covalently conjugated molecules and furthermore, for selective induction of apoptotic cell death in folate receptor expressing cancer cells. The tested carrier system displays potential for simultaneous delivery of several anticancer agents specifically to cancer cells also in vivo.

Impact of folate absorption and transport for nutrition and drug targeting

In this thesis, interactions of folic acid with tea and tea components at the level of intestinal absorption have been investigated. Firstly, the interaction between folic acid and tea as well as tea catechins was studied in vitro, using Caco-2 cell monolayers and secondly, a clinical trial was designed and carried out. In addition, targeting of folic acid conjugated nanoparticles to FR expressing Caco-2 cells was studied in order to evaluate the principle of nutrient-receptor-coupled transport for drug targeting. In the first part of this work, it was shown that EGCG and ECG (gallated catechins) inhibit folic acid uptake (IC50 of 34.8 and 30.8 µmol/L) comparable to MTX (methotrexate) under these experimental conditions. Moreover, commercial green and black tea extracts inhibited folic acid uptake with IC50 values of approximately 7.5 and 3.6 mg/mL, respectively. These results clearly indicate an interaction between folic acid and green tea catechins at the level of intestinal uptake. The mechanism responsible for the inhibition process might be the inhibition of the influx transport routes for folates such as via RFC and/or PCFT. For understanding the in vivo relevance of this in vitro interaction, a phase one, open-labeled, randomized, cross-over clinical study in seven healthy volunteers was designed. For the 0.4 mg folic acid dose, the mean Cmax decreased by 39.2% and 38.6% and the mean AUC0 decreased by 26.6% and 17.9% by green tea and black tea, respectively. For the 5 mg folic acid dose, the mean Cmax decreased by 27.4% and mean AUC0 decreased by 39.9% when taken with green tea. The results of the clinical study confirm the interaction between tea and folic acid in vivo leading to lower bioavailabilities of folic acid. In the second part of the thesis, targeting studies using folic acid conjugated nanoparticles were conducted. Folic acid conjugated nanoparticles were shown to be internalized by the cell via FR (folate receptor) mediated endocytosis. DNA block copolymer micelles equipped with 2, 11 and 28 folic acid units respectively were applied on FR expressing Caco-2 cells. There was a direct proportion in the amount of internalized nanoparticle and the number of folic acid units on the periphery of the nanoparticle. To sum up, throughout this thesis, the importance of folic acid for nutrition and nutrient and drug related interactions of folic acid at intestinal level was shown. Furthermore, significance of FRs in targeting for cancer chemotherapy was demonstrated in in vitro cell culture experiments. Folic acid conjugated DNA block copolymer micelles were suggested as efficient nanoparticles for targeted drug delivery.

Chemosensitization of carcinoma cells using epithelial cell adhesion molecule-targeted liposomal antisense against bcl-2/bcl-xL

Nanoscale drug delivery systems, such as sterically stabilized immunoliposomes binding to internalizing tumor-associated antigens, can increase therapeutic efficacy and reduce toxicity to normal tissues compared with nontargeted liposomes. The epithelial cell adhesion molecule (EpCAM) is of interest as a ligand for targeted drug delivery because it is abundantly expressed in solid tumors but shows limited distribution in normal tissues. To generate EpCAM-specific immunoliposomes for targeted cancer therapy, the humanized single-chain Fv antibody fragment 4D5MOCB was covalently linked to the exterior of coated cationic liposomes. As anticancer agent, we encapsulated the previously described antisense oligonucleotide 4625 specific for both bcl-2 and bcl-xL. The EpCAM-targeted immunoliposomes (SIL25) showed specific binding to EpCAM-overexpressing tumor cells, with a 10- to 20-fold increase in binding compared with nontargeted control liposomes. No enhanced binding was observed on EpCAM-negative control cells. On cell binding, SIL25 was efficiently internalized by receptor-mediated endocytosis, ultimately leading to down-regulation of both bcl-2 and bcl-xL expression on both the mRNA and protein level, which resulted in enhanced tumor cell apoptosis. In combination experiments, the use of SIL25 led to a 2- to 5-fold sensitization of EpCAM-positive tumor cells of diverse origin to death induction by doxorubicin. Our data show the promise of EpCAM-specific drug delivery systems, such as antisense-loaded immunoliposomes, for targeted cancer therapy.

Targeted Multistage Delivery of Nanoparticles to the Bone Marrow

Bone marrow is a target organ site involved in multiple diseases including myeloproliferative disorders and hematologic malignancies and metastases from breast and prostate. Most of these diseases are characterized with poor quality of life, and the treatment options are only palliative due to lack of delivery mechanisms for systemically injected drugs which results in dose limitation to protect the healthy hematopoietic cells. Therefore, there is a critical need to develop effective therapeutic strategies that allow for selective delivery of therapeutic payload to the bone marrow. Nanotechnology-based drug delivery systems provide the opportunity to deliver drugs to the target tissue while decreasing exposure to normal tissues. E-selectin is constitutively expressed on the bone marrow vasculature, but almost absent in normal vessels, and therefore, E-selectin targeted drug delivery presents an ideal strategy for the delivery of therapeutic nanoparticles to the bone marrow. The objective of this study was to develop a novel bone marrow targeted multistage vector (MSV) via E-selectin for delivery of therapeutics and imaging agents. To achieve this goal, Firstly, an E-selectin thioaptamer (ESTA) ligand was identified through a two-step screening from a combinatorial thioaptamer library. Next, ESTA-conjugated MSV (ESTA-MSV) were developed and evaluated for their stability and binding to E-selectin expressing endothelial cells. Different types of nanoparticles including liposomes, quantum dots, and iron oxide nanoparticles were loaded into the porous structure of ESTA-MSV. In vivo targeting experiments demonstrated 8-fold higher accumulation of ESTA-MSV in the mouse bone marrow as compared to non-targeted MSV Furthermore, intravenous injection of liposomes loaded ESTA-MSV resulted in a significantly higher accumulation of liposome in the bone marrow space as compared to injection of non-targeted MSV or liposomes alone. Overall this study provides first evidence that E-selectin targeted multistage vector preferentially targets to bone marrow vasculature and delivers larger amounts of nanoparticles. This delivery strategy holds potential for the selective delivery of large amounts of therapeutic payload to the vascular niches in the bone marrow for the treatment of bone marrow associated diseases.

Conjugation of an antibody to cross-linked fibrin for targeted delivery of anti-restenotic drugs

There is an urgent need to treat restenosis, a major complication of the treatment of arteries blocked by atherosclerotic plaque, using local delivery techniques. We observed that cross-linked fibrin (XLF) is deposited at the site of surgical injury of arteries. An antibody to XLF, conjugated to anti-restenotic agents, should deliver the drugs directly and only to the site of injury. An anti-XLF antibody (H93.7C.1D2/48; 1D2) was conjugated to heparin (using N-succinimidyl 3-(2-pyridyldithio)-propionate), low molecular weight heparin (LMWH) (adipic acid dihydrazide) and rapamycin (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysuccinimide), and the conjugates purified and tested for activity before use in vivo. Rabbits had their right carotid arteries de-endothelialised and then given a bolus of 1D2-heparin, 1D2-LMWH or 1D2-rapamycin conjugate or controls of saline, heparin, LMWH, rapamycin or 1D2 (+/-heparin bolus) and sacrificed after 2 or 4 weeks (12 groups, n=6/group). Rabbits given any of the conjugates had minimal neointimal development in injured arteries, with up to 59% fewer neointimal cells than those given control drugs. Rabbits given 1D2-heparin or 1D2-LMWH had an increased or insignificant reduction in luminal area, with positive remodelling, while the medial and total arterial areas of rabbits given 1D2-rapamycin were not affected by injury. Arteries exposed to 1D2-heparin or 1D2-rapamycin had more endothelial cells than rabbits given control drugs. Thus, XLF-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall, where the conjugates can influence remodelling, re-endothelialisation and neointimal cell density, with reduced neointimal formation. (C) 2004 Elsevier B.V. All rights reserved.

Targeted delivery of heparin and LMWH using a fibrin antibody prevents restenosis

This study investigates a stent-less local delivery system for anti-restenotic agents utilizing antibodies to cross-linked fibrin (XLF). Heparin and low molecular weight heparin (LMWH) were conjugated to an antibody to cross-linked fibrin D-dinner (1D2). Rabbit right carotid arteries were injured with a balloon catheter, then the animals were given a bolus injection of 40 mug/k,g 1D2-heparin (26-70 mug/kg heparin) or 1D2-LMWH (29-80 mug/kg LMWH) conjugates or controls of saline (0.5 ml/kg), heparin (150 U/kg), LMWH (2 mg), or 1D2 (40 mug/kg), with or without a heparin bolus and sacrificed after 2 weeks (8 groups, n = 6/group). The injured artery of rabbits given 1D2-heparin or 1D2-LMWH conjugates had reduced neointimal development, with decreased luminal narrowing and positive remodelling compared with animals given control drugs. Animals given 1D2-heparin conjugate (with a heparin bolus) had three to five times more endothelial cells than the rabbits given saline or unconjugated heparin, while rabbits given 1D2-LMWH conjugate had up to 59% fewer neointimal cells than those given unconjugated drugs. There was little difference in extracellular matrix organization or composition. Thus cross-linked fibrin-antibodies can site-deliver anti-restenotic agents to injured areas of the artery wall where they influence wall remodelling and endothelial and neointimal cell number, reducing neointimal formation without systemic complications. Local delivery of anti-restenotic agents should minimise systemic effects, bleeding complications and potentially the cost of treatment due to a single, lower dose. (C) 2004 Elsevier Ireland Ltd. All rights reserved.

Strategies for local drug delivery targeting the oesophagus

Localised, targeted drug delivery to the oesophagus offers the potential for more effective delivery and reduced drug dosages, coupled with increased patient compliance. This thesis considers bioadhesive liquids, orally retained tablets and films as well as chewable dosage forms as drug delivery systems to target the oesophagus. Miconazole nitrate was used as a model antifungal agent. Chitosan and xanthan gum hydrogels were evaluated as viscous polymer viables with the in vitro retention, drug release and minimum inhibitory concentration values of the formulations measured. Xanthan showed prolonged retention on the oesophageal surface in vitro yet chitosan reduced the MIC value; both polymers offer potential for local targeting to the oesophagus. Cellulose derivatives were investigated within orally retained dosage forms. Both drug and polymer dissolution rates were measured to investigate the drug release mechanism and to develop a formulation with concomitant drug and polymer release to target the oesophagus with solubilised drug within a viscous media. Several in vitro dissolution methods were evaluated to measure drug release from chewable dosage forms with both drug and polymer dissolution quantified to investigate the effects of dissolution apparatus on drug release. The results from this thesis show that a range of drug delivery strategies that can be used to target drug to the oesophagus. The composition of these formulations as well as the methodology used within the development are crucial to best understand the formulation and predict its performance in vivo.