Molecular release from a polymeric microreservoir device: Influence of chemistry, polymer swelling, and loading on device performance

A polymeric microreservoir device for controlled-release drug delivery relies on the degradation of thin poly(lactic-co-glycolic acid) membranes that seal each reservoir to achieve pulsatile drug delivery. In vitro release studies in which the swelling of the reservoir membranes was measured indicate a correlation between the release times of various radiolabeled molecules from the devices and the time at which the maximum membrane swelling was observed. Varying the chemistry (lipophilicity/hydrophilicity) or molecular weight of the molecules loaded into the devices did not appear to affect the degree of membrane swelling that was observed, or the time at which the molecules were released from the devices. The amount of drug that was loaded into the reservoirs also did not appear to affect the observed release time of the drug from the device, a significant departure from the behavior of many matrix-type polymeric drug delivery systems.     

Fabrication of multi-layered biodegradable drug delivery device based on micro-structuring of PLGA polymers

A programmable and biodegradable drug delivery device is desirable when a drug needs to be administered locally. While most local drug delivery devices made of biodegradable polymers relied on the degradation of the polymers, the degradation-based release control is often limited by the property of the polymers. Thus, we propose micro-geometry as an alternative measure of controlling drug release. The proposed devices consist of three functional layers: diffusion control layer via micro-orifices, diffusion layer, and drug reservoir layers. A micro-fabrication technology was used to shape an array of micro-orifices and micro-cavities in 85/15PLGA layers. A thin layer of fast degrading 50/50PLGA was placed as the diffusion layer between the 85/15PLGA layers to prevent any burst-type release. To modulate the release of the devices, the dimension and location of the micro-orifices were varied and the responding in vitro release response of tetracycline was monitored over 2 weeks. The release response to the different micro-geometry was prominent and further analyzed by FEM simulation. Comparison of the experiments to the simulated results identified that the variation of micro-geometry influenced also the volume-dependent degradation rate and induced the osmotic pressure.     

Co-effect of aqueous solubility of drugs and glycolide monomer on in vitro release rates from poly(D,L-lactide-co-glycolide) discs and polymer degradation

The objective of this study was to investigate the effect of aqueous solubility of model drugs and glycolide monomer (GM) from poly(D,L-lactide-co-glycolide) (PLGA) discs on in vitro release rates and polymer degradation. 5-Fluorouracil (5-FU), a water-soluble compound, and dexamethasone in a water-insoluble base form were selected as model drugs. Glycolide monomer, that has moderate solubility in water, was a non-toxic and biodegradable additive as a derivative material of hydrolysis of PLGA in order to obtain desirable drugs release rates. PLGA discs with or without GM were formulated by means of compression molding method. The prepared polymeric discs were incubated at 37 degrees C in phosphate-buffered saline (PBS, pH 7.4) and characterized at scheduled time points for water uptake, mass loss, diameter and morphology change, molecular weight and composition change using scanning electron microscopy (SEM), gel-permeation chromatography (GPC), and H-NMR, respectively. The supernatants were taken out of the sample vials and were analyzed for drug release. The 5-FU release was found to be increasing in proportion to the drug loading amount with an initial burst for 5 days, while dexamethasone release showed inverse relationship with the increasing drug loading amount. However, the release behaviors of 5-FU and dexamethasone polymeric discs containing GM showed faster release rates than control discs (without GM) and did not show lag periods during the in vitro release test due to adding GM, which acted as a channeling agent that has moderate solubility in water. Polymer degradation was found to be affected by aqueous solubility of drugs and GM. In conclusion, we observed that drugs release rates were influenced by their aqueous solubility and loading amount and also GM plays a major role in controlling drug release rates regardless of solubility of drugs. This system appears to be promising for controlled drug delivery aimed at local therapy.     

Degradation behaviour in vitro for poly(D,L-lactide-co-glycolide) as drug carrier

Biodegradable polymers have been extensively investigated because of regulating drug release rate easily, obviating the need to remove the device, and good biocompatibility. Among the biodegradable polymers currently under investigation, poly(D,L-lactide-co-glycolide) (PLGA) copolymers are the most widely studied because of their long history of safe clinical use as drug carrier. 50 : 50 PLGA was used as a model degradable polymer in this study to investigate the degradation behaviour on drug release from bulk degradable polymers in vitro. 5-fluorouracil (5-FU) was used as a model drug. Molecular weight change, residual mass, water uptake, morphological change of PLGA wafers, and pH of release test medium were characterized to investigate the effect of polymer degradation on drug release. The release rate of 5-FU increased with the increase of 5-FU loading amount and the release profiles of 5-FU irrespective of 5-FU loading amount followed near first order release kinetics.     

Fabrication and characterization of monodisperse PLGA–alginate core–shell microspheres with monodisperse size and homogeneous shells for controlled drug release

Monodisperse PLGA–alginate core–shell microspheres with controlled size and homogeneous shells were first fabricated using capillary microfluidic devices for the purpose of controlling drug release kinetics. Sizes of PLGA cores were readily controlled by the geometries of microfluidic devices and the fluid flow rates. PLGA microspheres with sizes ranging from 15 to 50 μm were fabricated to investigate the influence of the core size on the release kinetics. Rifampicin was loaded into both monodisperse PLGA microspheres and PLGA–alginate core–shell microspheres as a model drug for the release kinetics studies. The in vitro release of rifampicin showed that the PLGA core of all sizes exhibited sigmoid release patterns, although smaller PLGA cores had a higher release rate and a shorter lag phase. The shell could modulate the drug release kinetics as a buffer layer and a near-zero-order release pattern was observed when the drug release rate of the PLGA core was high enough. The biocompatibility of PLGA–alginate core–shell microspheres was assessed by MTT assay on L929 mouse fibroblasts cell line and no obvious cytotoxicity was found. This technique provides a convenient method to control the drug release kinetics of the PLGA microsphere by delicately controlling the microstructures. The obtained monodisperse PLGA–alginate core–shell microspheres with monodisperse size and homogeneous shells could be a promising device for controlled drug release.     

Hydrolytic degradation of tyrosine-derived polycarbonates, a class of new biomaterials. Part II: 3-yr study of polymeric devices

The kinetics and mechanisms of in vitro degradation of tyrosine-derived polycarbonates, a new class of polymeric biomaterials, were studied extensively at 37 degrees C. These polymers carry an alkyl ester pendent chain that allows the fine-tuning of the polymer's material properties, its biological interactions with cells and tissue, and its degradation behavior. The polymer carrying an ethyl ester pendent chain, poly(DTE carbonate), has been established as a promising orthopedic implant material, exhibiting bone apposition when in contact with hard tissue. Tyrosine-derived polycarbonates are relatively stable and degrade only very slowly in vitro. Therefore, accelerated studies were conducted at 50 and 65 degrees C to observe the behavior of polymers during the later stages of degradation. Varying the pendent chain length affected the rate of water uptake, initial degradation rate, and physical stability of the polymeric devices. During the 3-yr study, the polymer degraded by random chain cleavage of the carbonate bonds, accompanied by a relatively small amount of pendent chain de-esterification. No mass loss was observed during this period at 37 degrees C, but mass loss was readily evident during the accelerated studies at 50 and 65 degrees C. Thus, it is reasonable to assume that mass loss will occur also at 37 degrees C, albeit only after extensive backbone carbonate cleavage and pendent chain ester hydrolysis. The dimension and surface area of the devices influenced the initial degradation rate, but did not significantly affect the overall rate of degradation. No evidence of "acid dumping" or the release of acidic residues found during the degradation of poly(D,L-lactic acid) were observed for this family of tyrosine-derived polycarbonates.     

Morphological and degradation studies of sirolimus-containing poly(lactide-co-glycolide) discs

The effect of residual solvent and copolymer ratio on the in vitro degradation and drug release behavior of a bioabsorbable polymer/drug system was investigated in an effort to understand and develop the use of these excipients for controlled drug delivery devices. Sirolimus-containing poly(lactide-co-glycolide) (PLGA) discs were fabricated by a solution-casting method using dimethyl sulfoxide (DMSO) as the solvent. The residual DMSO was removed from a set of discs by supercritical carbon dioxide extraction, and reflections of crystalline sirolimus were observed in the wide-angle X-ray scattering profile observed after extraction. A correlation was not observed between the extent of drug crystallization and extraction conditions and copolymer ratio. Mass loss, molecular weight, and sirolimus release were monitored during an in vitro study of the oven-dried neat PLGA, sirolimus-containing PLGA, and extracted sirolimus-containing PLGA discs during 56 days. The sirolimus-containing PLGA discs with residual DMSO exhibited a faster sirolimus release rate compared to the extracted discs. The residual DMSO facilitated release of sirolimus. The discs that contained PLGA with higher glycolide content, particularly 50% glycolide, degraded faster and exhibited faster sirolimus release.     

Preparation, characterization, and in vitro evaluation of physostigmine-loaded poly(ortho ester) and poly(ortho ester)/poly(D,L-lactide-co-glycolide) blend microspheres fabricated by spray drying

The physostigmine-loaded poly(ortho ester) (POE), poly(dl-lactide-co-glycolide) (PLGA) and POE/PLGA blend microspheres were fabricated by a spray drying technique. The in vitro degradation of, and physostigmine release from, the microspheres were investigated. SEM analysis showed that the POE and POE/PLGA blend particles were spherical. They were better dispersed when compared to the pure PLGA microspheres. Two glass transition temperature ( Tg ) values of the POE/PLGA blend microspheres were observed due to the phase separation of POE and PLGA in the blend system. XPS analysis proved that POE dominated the surfaces of POE/PLGA blend microspheres, indicating that the blend microspheres were coated with POE. The encapsulation efficiencies of all the microspheres were more than 95%. The incorporation of physostigmine reduced the Tg value of microspheres. The Tg value of the degrading microspheres increased with the release of physostigmine. For instance, POE blank microspheres and physostigmine-loaded POE microspheres had a Tg value of 67 degrees C and 48 degrees C, respectively. After 19 days in vitro incubation, Tg of the degrading POE microspheres increased to 55 degrees C. Weight loss studies showed that the degradation of the blend microspheres was accelerated with the presence of PLGA because its degradation products catalyzed the degradation of both POE and PLGA. The release rate of physostigmine increased with increase of PLGA content in the blend microspheres. The initial burst release of physostigmine was effectively suppressed by introducing POE to the blend microspheres. However, there was an optimized weight ratio of POE to PLGA (85:15 in weight), below which a high initial burst was induced. The POE/PLGA blend microspheres may make a good drug delivery system.     

Degradation of electrospun PLGA-chitosan/PVA membranes and their cytocompatibility in vitro

Nanofibrious composite poly(lactide-co-glycolide) (PLGA) and chitosan/poly(vinyl alcohol) (PVA) membranes were prepared by simultaneously electrospinning PLGA and chitosan/PVA from two different syringes. The in vitro degradation of PLGA and cross-linked composite membranes was examined for up to 10 weeks in phosphate-buffered saline (PBS, pH 7.4) at 37 degrees C. The pH of PBS, the weight average molecular weight of PLGA, fiber morphology and mechanical properties, including tensile strength, Young's modulus and elongation-at-break, were measured as a function of degradation time. The fibrous composite membranes were further investigated as a promising scaffold for human embryo skin fibroblasts (hESFs) culture. The cell adhesion and morphology of hESFs seeded on each electrospun membrane was observed using scanning electron microscope and inverted phase contrast microscopy after Wright-Giemsa staining. The introduction of chitosan/PVA component changed the hydrophilic/hydrophobic balance and, thus, influenced degradation behavior and mechanical properties of the composite membranes during degradation. The cells could not only favorably attach and grow well on the composite membranes, but were also able to migrate and infiltrate the membranes. Therefore, the results suggest that the composite membranes can positively mimic the structure of natural extracellular matrices and have the potential for application as three-dimensional tissue-engineering scaffolds.     

Evaluation of in vitro drug release, pH change, and molecular weight degradation of poly(L-lactic acid) and poly(D,L-lactide-co-glycolide) fibers

Biodegradable fibers of poly(L-lactic acid) (PLLA) and poly(D,L-lactide-co-glycolide) (PLGA) that encapsulated a water-soluble drug were created by a patented technique consisting of wet-spinning a water-in-oil emulsion. These fibers are 2.4% by mass drug, which is slowly released, making these fibers potential candidates for implantation as drug delivery devices and/or tissue-engineering substrates. Drug release kinetics and changes in molecular weight were investigated over time. This study demonstrated that drug release rates and molecular weight degradation are a function of the amount of aqueous phase added as an emulsion during fabrication. The type of polymer used (PLLA or PLGA) determines the molecular weight degradation rates, but has little effect on drug release kinetics.     

Molecular weight distribution changes during degradation and release of PLGA nanoparticles containing epirubicin HCl

The molecular dynamics of the degradation process of poly(lactic-co-glycolic acid) nanospheres were investigated during the degradation process usually observed when these polymers are used as controlled release carriers. The molecular weight distribution of PLGA samples was determined over a period of 32 days by accurately analyzing the molecular weight distribution of the polymer as a function of time as degradation progressed. The molecular weight distribution shifted gradually to lower average molecular weights over 32 days, with significantly smaller molecular weight components appearing at 8-12 days. In addition, the degradation of nanospheres containing epirubicin HCI was analyzed and increasing the amount of epirubicin from 1.7 to 3.4 to 6.7 wt% was found to hasten the degradation of the nanoparticles and subsequently affect the release behavior from these particles. This is believed to be the first time that such molecular dynamics have been presented for the degradation of PLGA nanoparticle formulations containing a drug for controlled delivery.     

Release of Hydrophilic Drug from Biodegradable Polymer Blends

This paper reports on the release behavior of the drug lidocaine-HCl from a immiscible polymeric blend. Biodegradable polymer blends of poly(L-lactic acid)/poly(lactic-co-glycolic acid) (PLLA/PLGA) were loaded with lidocaine-HCl, and the release of lidocaine-HCl from these blends was monitored. It was found that the release profiles were significantly affected by the affinity and subsequent partitioning of the drug into one of the two phases in the blends. It was hypothesized that the hydrophilic lidocaine-HCl seems to have a tendency to reside in the PLGA component of the PLLA/PLGA blend. This resulted in a release very much controlled by the degradation of PLGA, even when PLLA is the major phase of the blend. A mathematical model was further employed to quantify the partitioning, as well as model the lidocaine-HCl release profiles of different blend compositions.     

Differential degradation rates in vivo and in vitro of biocompatible poly(lactic acid) and poly(glycolic acid) homo- and co-polymers for a polymeric drug-delivery microchip

The biocompatibility and biodegradation rate of component materials are critical when designing a drug-delivery device. The degradation products and rate of degradation may play important roles in determining the local cellular response to the implanted material. In this study, we investigated the biocompatibility and relative biodegradation rates of PLA, PGA and two poly(lactic-co-glycolic acid) (PLGA) polymers of 50:50 mol ratio, thin-film component materials of a drug-delivery microchip developed in our laboratory. The in vivo biocompatibility and both in vivo and in vitro degradation of these materials were characterized using several techniques. Total leukocyte concentration measurements showed normal acute and chronic inflammatory responses to the PGA and low-molecular-weight PLGA that resolved by 21 days, while the normal inflammatory responses to the PLA and high-molecular-weight PLGA were resolved but at slower rates up to 21 days. These results were paralleled by thickness measurements of fibrous capsules surrounding the implants, which showed greater maturation of the capsules for the more rapidly degrading materials after 21 days, but less mature capsules of sustained thicknesses for the PLA and high-molecular-weight PLGA up to 49 days. Gel-permeation chromatography of residual polymer samples confirmed classification of the materials as rapidly or slowly degrading. These materials showed thinner fibrous capsules than have been reported for other materials by our laboratory and have suitable biocompatibility and biodegradation rates for an implantable drug-delivery device.     

Biodegradable poly(polyol sebacate) polymers

We have developed a family of synthetic biodegradable polymers that are composed of structural units endogenous to the human metabolism, designated poly(polyol sebacate) (PPS) polymers. Material properties of PPS polymers can be tuned by altering the polyol monomer and reacting stiochiometric ratio of sebacic acid. These thermoset networks exhibited tensile Young's moduli ranging from 0.37+/-0.08 to 378+/-33 MPa with maximum elongations at break from 10.90+/-1.37% to 205.16+/-55.76%, and glass transition temperatures ranging from approximately 7-46 degrees C. In vitro degradation under physiological conditions was slower than in vivo degradation rates observed for some PPS polymers. PPS polymers demonstrated similar in vitro and in vivo biocompatibility compared to poly(L-lactic-co-glycolic acid) (PLGA).     

Synthesis, characterization, biodegradation, and drug delivery application of biodegradable lactic/glycolic acid polymers: Part III. Drug delivery application

Lactic/glycolic acid polymers (PLGA) are widely used for drug delivery systems. The microsphere formulation is the most interesting dosage form of the PLGA-based controlled release devices. In this study, the previously reported PLGA were used to prepare drug-containing microspheres. Progesterone was used as a model drug. The progesterone microspheres were prepared from PLGA having varied compositions and varied molecular weight. The microscopic characterization shows that the microspheres are spherical, nonaggregated particles. The progesterone-containing PLGA microspheres possess a Gaussian size distribution, having average size from 70-134 microm. A solvent extraction method was employed to prepare the microspheres. The microencapsulation method used in this study has high drug encapsulation efficiency. The progesterone release from the PLGA microspheres and the factors affecting the drug release were studied. The release of progesterone from the PLGA microspheres is affected by the properties of the polymer used. The drug release is more rapid from the microspheres prepared using the PLGA having higher fraction of glycolic acid moiety. The drug release from the microspheres composed of higher molecular weight PLGA is faster. The drug content in microspheres also has an effect on the drug release. Higher progesterone content in microspheres yields a quicker initial burst release of the drug.     

In vitro study of anticancer drug doxorubicin in PLGA-based microparticles

Doxorubicin (DOX), also known as adriamycin, is an anthracycline drug commonly used in cancer chemotherapy. Unfortunately, its therapeutic potential has been restricted by its dose limited cardiotoxicity and the resistance developed by the tumor cells to the molecule after some time of treatment. One way to overcome these problems is to encapsulate the drug in poly (D, L-lactide-co-glycolide) (PLGA) microparticles. This paper investigates the release characteristics of DOX from polymeric carriers fabricated using the spray-drying technique. The encapsulation efficiency, size and morphology of the various polymeric devices were also determined. In order to improve the release characteristics, Pluronic P105 (PLU) and poly (L-Lactide) (PLLA) are individually used in combination with PLGA. Finally, a cytotoxicity test was performed using Glioma C6 cancer cells to investigate the cytotoxicity of DOX delivered from PLGA microparticles. It has been found that the cytotoxicity of DOX to Glioma C6 cancer cells is enhanced when DOX is delivered from PLGA polymeric carrier.     

Incorporation and in vitro release of doxorubicin in thermally sensitive micelles made from poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide)-b-poly(D,L-lactide-co-glycolide) with varying compositions

Thermally sensitive block copolymers, poly(N-isopropylacrylamide-co-N, N-dimethylacrylamide)-b-poly(d,l-lactide-co-glycolide) [P(NIPAAm-co-DMAAm)-b-poly(D,L-lactide-co-glycolide) (PLGA)] with different compositions and lengths of PLGA block are synthesized and utilized to fabricate micelles containing doxorubicin (DOX), a model anticancer drug, by a membrane dialysis method for targeted anticancer drug delivery. The critical association concentration (CAC) of the polymers ranges from 4.0 to 25.0 mg/L. An increased length of core-forming block PLGA leads to a decrease in the CAC. The clearly defined core-shell structure of micelles is proved by 1H-NMR analyses of the micelles in CDCl3 and D2O. The morphology of the micelles is analyzed by transmission electron microscopy, showing a spherical structure of both blank and drug-loaded micelles. The results obtained from dynamic light scattering show that the blank and drug-loaded micelles have an average size below 200 nm. The lower critical solution temperature (LCST) of the micelles made from the various polymers is similar, around 39 degrees C in phophate-buffered solution (PBS). The presence of serum in PBS does not alter the LCST significantly. The drug loading capacity varies depending on the PLGA block. The polymers are degradable, and the degradation of PLGA-based polymers is faster than that of poly(lactide) (PLA)-based polymer. The DOX-loaded micelles are stable in PBS containing serum at 37 degrees C but deform at 39.5 degrees C above the normal body temperature, thus triggering DOX release. It is revealed by confocal laser scanning microscopy that free DOX molecules enter cell nuclei very fast and DOX-loaded micelles accumulate mostly in cytoplasm after endocytosis. At a temperature above the LCST, more DOX molecules release from the micelles and enter the nuclei as compared to the temperature below the LCST. DOX-loaded micelles show greater cytotoxicity at a temperature above the LCST. The P(NIPAAm-co-DMAAm)-b-PLGA micelles developed may be a good carrier for anticancer drug delivery.     

Delivering neuroactive molecules from biodegradable microspheres for application in central nervous system disorders

Nerve growth factor (NGF) may enhance axonal regeneration following injury to the central nervous system (CNS), such as after spinal cord injury. The release profile of NGF, co-encapsulated with ovalbumin, was tailored from biodegradable polymeric microspheres using both polymer degradation and protein loading. Biodegradable polymeric microspheres were prepared from PLGA 50/50, PLGA 85/15, PCL and a blend of PCL/PLGA 50/50 (1:1, w/w), where the latter was used to further tailor the degradation rate. The amount of protein loaded in the microspheres was varied, with PCL encapsulating the greatest amount of protein and PLGA 50/50 encapsulating the least. A two-phase release profile was observed for all polymers where the first phase resulted from release of surface proteins and the second phase resulted predominantly from polymer degradation. Polymer degradation influenced the release profile most notably from PLGA 50/50 and PLGA 85/15 microspheres. The amount and bioactivity of released NGF was followed over a 91 d period using a NGF-ELISA and PC12 cells, respectively. NGF was found to be bioactive for 91 d, which is longer than previously reported.     

A novel polyethylene depot device for the study of PLGA and P(FASA) microspheres in vitro and in vivo

Polymer microspheres (0.5-5.0 microm) are difficult to characterize in vivo because they degrade, migrate, and are endocytosed. A novel polyethylene mesh pouch containing microspheres allowed for retrieval of degraded polymeric products from rats without affecting the rate of degradation. Pouches containing poly(lactic-co-glycolic acid) (PLGA) or poly(fumaric-co-sebacic acid) (P(FASA)) microspheres were implanted intramuscularly, subcutaneously, and intraperitoneally and analyzed after 3, 7, 14, and 28 days. In vivo, subcutaneous or intraperitoneal implants experienced an immediate mass loss and a delayed decrease in molecular weight (Mw). Intramuscular implants behaved similarly to in vitro samples, decreasing in Mw immediately and lagging in mass loss. These results suggest that mass loss, which is usually dependent on Mw loss in vitro, may be directly due to enzymatic, rather than hydrolytic, degradation subcutaneously and intraperitoneally, while intramuscular implants appear to be mostly dependent on hydrolytic cleavage. This observation is further supported by histology. Additional experiments on pouches loaded with PLGA microspheres encapsulating osteoprotegerin, a protein drug used to prevent bone resorption, revealed that use of the device prevented the artifactual polymer compression inherent to microsphere centrifugation during release studies and allowed for the extraction of active protein from microspheres implanted for 3 days in vivo.     

The effect of polyethylene glycol structure on paclitaxel drug release and mechanical properties of PLGA thin films

Thin films of poly(lactic acid-co-glycolic acid) (PLGA) incorporating paclitaxel typically have slow release rates of paclitaxel of the order of 1 μg day(-1) cm(-2). For implementation as medical devices a range of zero order release rates (i.e. 1-15 μg day(-1) cm(-2)) is desirable for different tissues and pathologies. Eight and 35 kDa molecular weight polyethylene glycol (PEG) was incorporated at 15%, 25% and 50% weight ratios into PLGA containing 10 wt.% paclitaxel. The mechanical properties were assessed for potential use as medical implants and the rates of release of paclitaxel were quantified as per cent release and the more clinically useful rate of release in μg day(-1) cm(-2). Paclitaxel quantitation was correlated with the release of PEG from PLGA, to further understand its role in paclitaxel/PLGA release modulation. PEG release was found to correlate with paclitaxel release and the level of crystallinity of the PEG in the PLGA film, as measured by Raman spectrometry. This supports the concept of using a phase separating, partitioning compound to increase the release rates of hydrophobic drugs such as paclitaxel from PLGA films, where paclitaxel is normally homogeneously distributed/dissolved. Two formulations are promising for medical device thin films, when optimized for tensile strength, elongation, and drug release. For slow rates of paclitaxel release an average of 3.8 μg day(-1) cm(-2) using 15% 35k PEG for >30 days was achieved, while a high rate of drug release of 12 μg day(-1) cm(-2) was maintained using 25% 8 kDa PEG for up to 12 days.